Peralkylated oligopeptide mixtures

ABSTRACT

Linear C 1  -C 7  -alkyl peralkylated oligopeptide sets of molecules are disclosed, as are their methods of synthesis and use in acceptor binding assays. Each molecule or chain of a set contains the same number of two to about ten substituted C 1  -C 7  -alkyl peralkylated amino acid residues, and the member chains of a set are present in equimolar amounts. The chains of a set contain one or more predetermined peralkylated amino acid residues at one or more predetermined positions of the peralkylated oligopeptide chain. The set contains equimolar amounts of at least six different peralkylated amino acid residues at one or more of the same predetermined positions of the peralkylated oligopeptide chain. Libraries of such sets, processes for their use and solid support-linked peralkylated sets are also contemplated, as are specific permethylated oligopeptides.

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 08/257,782, filed Jun. 9,1994, now U.S. Pat. No. 5,480,971, which is a continuation-in-part ofapplication Ser. No. 08/079,144, filed Jun. 17, 1993, whose disclosuresare incorporated by reference.

TECHNICAL FIELD

The present invention relates to the synthesis and use of peptide-likemixtures. More particularly, the invention relates to the synthesis anduse of a mixture of peralkylated peptides whose peptide bond amidonitrogen atoms are alkylated as can be reactive hydrogens on side chainsand a N-terminal amino group, when present, and a C-terminal carboxylgroup.

BACKGROUND AND RELATED ART

Over the last several years, developments in peptide synthesistechnology have resulted in automated synthesis of peptides accomplishedthrough the use of solid phase synthesis methods. The solid phasesynthesis chemistry that made this technology possible was firstdescribed in Merrifield et al. J. Amer. Chem. Soc., 85:2149-2154 (1963).The "Merrifield method" has for the most part remained largely unchangedand is used in nearly all automated peptide synthesizers availabletoday.

In brief, the Merrifield method involves synthesis of a peptide chain onsolid support resin particles. These particles typically are comprisedof polystyrene cross-linked with divinyl benzene to form porous beadsthat are insoluble in both water and various organic solvents used inthe synthesis protocol. The resin particles contain a fixed amount ofamino- or hydroxylmethyl aromatic moiety that serves as the linkagepoint for the first amino acid in the peptide.

Attachment of the first amino acid entails chemically reacting itscarboxyl-terminal (C-terminal) end with derivatized resin to form thecarboxyl-terminal end of the oligopeptide. The alpha-amino end of theamino acid is typically blocked with a t-butoxy-carbonyl group (t-BOC)or with a 9-fluorenylmethyloxycarbonyl (Fmoc) group to prevent the aminogroup that could otherwise react from participating in the couplingreaction. The side chain groups of the amino acids, if reactive, arealso blocked (or protected) by various benzyl-derived protecting groupsin the form of ethers, thioethers, esters, and carbamates, andt-butyl-derived blockers for Fmoc syntheses.

The next step and subsequent repetitive cycles involve deblocking theamino-terminal (N-terminal) resin-bound amino acid (or terminal residueof the peptide chain) to remove the alpha-amino blocking group, followedby chemical addition (coupling) of the next blocked amino acid. Thisprocess is repeated for however many cycles are necessary to synthesizethe entire peptide chain of interest. After each of the coupling anddeblocking steps, the resin-bound peptide is thoroughly washed to removeany residual reactants before proceeding to the next. The solid supportparticles facilitate removal of reagents at any given step as the resinand resin-bound peptide can be readily filtered and washed while beingheld in a column or device with porous openings such as a filter.

Synthesized peptides are released from the resin by acid catalysis(typically with hydrofluoric acid or trifluoroacetic acid), whichcleaves the peptide from the resin leaving an amide or carboxyl group onits C-terminal amino acid. Acidolytic cleavage also serves to remove theprotecting groups from the side chains of the amino acids in thesynthesized peptide. Finished peptides can then be purified by any oneof a variety of chromatography methods.

Though most peptides are synthesized with the above described procedureusing automated instruments, a recent advance in the solid phase methodby R. A. Houghten allows for synthesis of multiple independent peptidessimultaneously through manually performed means. The "SimultaneousMultiple Peptide Synthesis" ("SMPS") process is described in U.S. Pat.No. 4,631,211 (1986); Houghten, Proc. Natl. Acad. Sci., 82:5131-5135(1985); Houghten et al., Int. J. Peptide Protein Res., 27:673-678(1986); Houghten et al., Biotechniques, 4, 6, 522-528 (1986), andHoughten, U.S. Pat. No. 4,631,211, whose disclosures are incorporated byreference.

Illustratively, the SMPS process employs porous containers such asplastic mesh bags to hold the solid support synthesis resin. AMerrifield-type solid-phase procedure is carried out with theresin-containing bags grouped together appropriately at any given stepfor addition of the same, desired amino acid residue. The bags are thenwashed, separated and regrouped for addition of subsequent same ordifferent amino acid residues until peptides of the intended length andsequence have been synthesized on the separate resins within eachrespective bag.

That method allows multiple, but separate, peptides to be synthesized atone time, since the peptide-linked resins are maintained in theirseparate bags throughout the process. The SMPS method has been used tosynthesize as many as 200 separate peptides by a single technician in aslittle as two weeks, a rate vastly exceeding the output of mostautomated peptide synthesizers.

A robotic device for automated multiple peptide synthesis has beenrecently commercialized. The device performs the sequential steps ofmultiple, separate solid phase peptide synthesis through iterativemechanical-intensive means. This instrument can synthesize up to 96separate peptides at one time, but is limited at present by the quantityof its peptide yield.

The interest in obtaining biologically active peptides forpharmaceutical, diagnostic and other uses would make desirable aprocedure designed to find a mixture of peptides or a single peptidewithin a mixture with optimal activity for a target application.Screening mixtures of peptides enables the researcher to greatlysimplify the search for useful therapeutic or diagnostic peptidecompounds. Mixtures containing hundreds of thousands or more peptidesare readily screened since many biochemical, biological and small animalassays are sensitive enough to detect activity of compounds that havebeen diluted down to the nanogram or even picogram per milliliter range,the concentration range at which naturally occurring biological signalssuch as peptides and proteins operate.

Almost all of the broad diversity of biologically relevantligand-receptor (or affector-acceptor) interactions occur in thepresence of a complex milieu of other substances (i.e., proteins make upapproximately 5-10 percent of plasma, e.g. albumin 1-3 percent,antibodies 2-5 percent-salts, lipids/fats, etc.). This is true forvirtually all biologically active compounds because most are commonlypresent, and active, at nanomolar and lower concentrations. Thesecompounds are also, in most instances, produced distant from theiraffection sites.

That a small peptide (or other molecule) can readily "find" an acceptorsystem, bind to it, and affect a necessary biological function prior tobeing cleared from the circulation or degraded suggests that a singlespecific peptide sequence can be present in a very wide diversity, andconcentration, of other individual peptides and still be recognized byits particular acceptor system (antibody, cellular receptor, etc.). Ifone could devise a means to prepare and screen a synthetic combinatoriallibrary of peptides, then the normal exquisite selectivity of biologicalaffector/acceptor systems could be used to screen through vast numbersof synthetic oligopeptides.

Of interest in screening very large numbers of peptides is work byGeysen et al., which deals with methods for synthesizing peptides withspecific sequences of amino acids and then using those peptides toidentify reactions with various receptors. See U.S. Pat. Nos. 4,708,871,4,833,092 and 5,194,392; P.C.T. Publications Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002(1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985);Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysenet al., J. Immunol.

Meth., 102:259-274 (1987); and Schoofs et al., J. Immunol., 140:611-616(1988).

In U.S. Pat. No. 5,194,392, Geysen describes a method for determiningso-called "mimotopes". A mimotope is defined as a catamer (a polymer ofprecisely defined sequence formed by the condensation of a precisenumber of small molecules), which in at least one of its conformationshas a surface region with the equivalent molecule topology to theepitope of which it is a mimic. An epitope is defined as the surface ofan antigenic molecule which is delineated by the area of interactionwith an antibody molecule.

The mimotopes are synthesized on a series of solid polymer (e.g.polyethylene with a coating of grafted polyacrylic acid) rods having adiameter of about 4 mm and a length of about 50 mm. A spacer formed byreaction of the ε-amino group of t-BOC-lysine methyl ester and thent-BOC-alanine was added to the grafted polyacrylic acid resins, followedby removal of the t-BOC group to provide an amino group to be used tobegin the syntheses.

A mixture of blocked (N-protected) amino acids containing differentamounts of each of the blocked (N-protected) twenty amino acids to beused was dissolved in dimethyl formamide and then coupled to the rods.That first coupling was repeated three times using conventional solidphase synthesis techniques. Twenty amino acid residues were individuallynext added to different rods so that twenty rod-linked 5-mer peptidesequences were prepared. Each sequence had a single, known amino acidresidue at the amino-terminus and an alleged equimolar mixture of aminoacid residues at each of the four other positions of the chain. Each ofthose twenty rod-linked peptides was then individually reacted with eachof the twenty amino acid residues to form 400 (20×20) rod-linked 6-merpeptides having the two amino-terminal positions defined and the fourremaining positions as mixtures. Two more positions of alleged equimolarmixtures of amino acids were then added, and the terminal amineacetylated to form N-acetyl 8-mers linked to the rods whose first twoamino acid positions were undefined (mixtures), followed by two definedpositions, followed by four undefined positions (mixtures), followed bythe spacer and then the supporting rods.

The 400 rod-linked N-acetyl 8-mer peptide mixture preparations were thenscreened in an ELISA assay using a monoclonal antibody to a desiredantigenic protein. The 8-mers having the preferential binding to theantibody were identified. Two sets of further 8-mers that contained theidentified best-binding 2-mer sequences within those 8-mers wereprepared.

A first set contained mixed amino acids at the three C-terminalpositions, followed toward the N-terminus, by a position containing eachof the twenty amino acids made by twenty separate couplings, theidentified 2-mer sequences, two further mixtures at the next twopositions, and an N-terminal acetyl group. The second group containedmixed amino acids at the four C-terminal positions, the identified 2-mersequences, a position made by separate couplings of each of the twentyamino acids, mixed amino acids as the terminal residues and anN-terminal acetyl group.

Each of those rod-linked N-acetyl 8-mers was again screened in an ELISAwith the monoclonal antibody. The preferential binding sequences foreach group were identified, and thus 4-mer, preferential-bindingsequences were identified.

The above process of separately adding each of the amino acids on eitherside of identified preferential-binding sequences was repeated until anoptimum binding sequence was identified.

The above method, although elegant, suffers from several disadvantagesas to peptides. First, owing to the small size of each rod used,relatively small amounts of each peptide is produced. Second, each assayis carried out using the rod-linked peptides, rather than the freepeptides in solution. Third, even though specific amounts of eachblocked amino acid are used to prepare the mixed amino acid residues atthe desired positions, there is no way of ascertaining that an equimolaramount of each residue is truly present at those positions.

Indeed, U.S. Pat. No. 5,194,392 contains a table of specific amounts ofeach N-protected amino acid to use to provide alleged equimolarity. Theprosecution history of that patent provides a revised table withdifferent amounts of N-protected amino acids for use.

Rutter et al. U.S. Pat. No. 5,010,175 discloses the preparation ofpeptide mixtures that are said to contain equimolar amounts of eachreacted amino acid at predetermined positions of the peptide chain.Those mixtures are also said to contain each peptide in retrievable andanalyzable amounts and are constructed by reacting mixtures of activatedamino acids in concentrations based on the relative coupling constantsof those activated amino acids.

The mixture of amino acids used for syntheses of peptides havingequimolar amounts of each residue is prepared by adjusting theconcentration of each amino acid in the reaction solution based on itsrelative coupling constant. Those relative coupling constants weredetermined by completely reacting the twenty naturally occurringresin-linked amino acids with each of the same twenty amino acids. Theseparate 400 resulting dipeptides were severed from their resins and theamount of each amino acid that coupled was determined.

Upon determining those 400 amounts, the 400 corresponding relative rateconstants were determined. The concentrations of the reactants were thanadjusted to obtain equimolarity of coupling using an algorithm said tobe not straightforward to calculate so that the affects of thepreviously bonded residue (acceptor) on the incoming amino acid can betaken into account.

In practice, acceptors of similar reactivities are reacted withappropriate mixtures of amino acids to achieve the desired results. Theconcentrations of reactants amino acids are then adjusted based on thecondensation results obtained. Acceptors of differing coupling rateswere said to be used in separate reaction mixtures.

U.S. Pat. No. 5,010,175 describes preparation of several pentapeptidessaid to have a single residue at one or more positions and mixtures offour residues at other positions. The mixed positions were reported tocontain their mixed residues at equimolarity plus-or-minus (±) about 20to about 24 percent.

A study using a mixture of the N-protected naturally occurring aminoacids was also reported. The amounts of N-protected amino acids usedwere based on their relative rate determinations, and adjusted toapproximate first-order kinetics by having each amino acid in at least10-fold excess over its final product. Relative rates were determined byaveraging values from the 400 separate reactions and additional data notprovided. A table of amounts of each of the twenty N-protected naturallyoccurring amino acids said to provide equimolarity when used as amixture is also provided in this patent.

In addition, Furka et al., (1988, 14th International Congress ofBiochemistry, Volume 5, Abstract FR:013) and (1988, Xth InternationalSymposium on Medicinal Chemistry, Budapest, Abstract 288, p. 168)described the synthesis of nine tetrapeptides each of which contained asingle residue at each of the amino- and carboxy-termini and mixtures ofthree residues at each position therebetween. These mixture positionswere obtained by physically mixing resins reacted with single aminoacids. The abstract further asserts that those authors' experimentsindicated that a mixture containing up to 180 pentapeptides could beeasily synthesized in a single run. No biological assays were reported.More recently, Furka et al., Int. J. Peptide Protein Res., 37:487-493(1991) reported on the synthesis of mixtures of 27 tetrapeptides and 180pentapeptides prepared by physically mixing reacted resin-linkedpeptides. Those peptides were synthesized with one or mixtures of threeor four residues at each position along the chain. No biological resultsusing those relatively simple mixtures were reported.

More recently, Huebner et al. U.S. Pat. No. 5,182,366 describedsubstantially the same process. Huebner et al. data provided for amixture of tetramers having a glycine at position 2 from the amino- (N-)terminus and each of five different amino acid residues at positions 1,3 and 4 from the N-terminus indicated that each of the residues atpositions 1, 3 and 4 were present in substantially equimolar amounts andthat glycine was present in its predicted amount. Similar data were alsoprovided for twenty-five groups of pentamers, each of which had twoknown residues at the amino-termini and mixtures of five residues eachat the remaining positions. No data were presented as to biologicalactivity or actually obtaining any selected peptide from the preparedmixtures.

A similar approach was also reported by Lam et al., Letters to Nature,354:82-84 (1991). Those workers reported the preparation of millions ofbead-linked peptides, each bead being said to contain a single peptide.The peptide-linked beads were reacted with a fluorescent- orenzyme-labeled acceptor. The beads bound by the acceptor were noted bythe label and were physically removed. The sequence of the bound peptidewas analyzed.

Recent reports (Devlin et al., Science, 249:404-405 1990! and Scott etal., Science, 249:386-390 1990!) have described the use of recombinantDNA and bacterial expression to create highly complex mixtures ofpeptides. More recently, Fodor et al., Science, 251:767-773 (1991),described the solid phase synthesis of thousands of peptides ornucleotides on glass microscope slides treated withaminopropyltriethoxysilane to provide amine functional groups.Predetermined amino acids were then coupled to predefined areas of theslides by the use of photomasks. The photolabile protecting group NVOC(nitroveratryloxycarbonyl) was used as the amino-terminal protectinggroup.

By using irradiation, a photolabile protecting group and masking, Fodoret al. reported preparation of an array of 1024 different peptidescoupled to the slide in ten steps. Immunoreaction with afluorescent-labeled monoclonal antibody was assayed with epifluorescencemicroscopy.

This elegant method is also limited by the small amount of peptide oroligonucleotide produced, by use of the synthesized peptide ornucleotide affixed to the slide, and also by the resolution of thephotomasks. This method is also less useful where the epitope bound bythe antibody is unknown because all of the possible sequences are notprepared.

The primary limitation of the above new approaches for the circumventionof individual screening of millions of individual peptides by the use ofa combinatorial library is the inability of the peptides generated inthose systems to interact in a "normal" manner with acceptor sites,analogous to natural interaction processes (i.e., free in solution at aconcentration relevant to the receptors, antibody binding sites, enzymebinding pockets, or the like being studied without the exclusion of alarge percentage of the possible combinatorial library), as well as thedifficulties inherent in locating one or more active peptides.Secondarily, the expression vector systems do not readily permit theincorporation of the D-forms of the natural amino acids or the widevariety of unnatural amine acids which would be of interest in the studyor development of such interactions.

Houghten et al., Letters to Nature, 354:84-86 (1991) reported use ofphysical mixtures in a somewhat different approach from those of Furkaet al., Huebner et al. and Lam et al., supra, by using solutions offree, rather than support-coupled, peptide libraries or sets thatovercomes several of the problems inherent in the above art. Here, 324exemplary hexamer mixtures that contained more than 34 million peptideswere first prepared whose N-terminal two positions were predeterminedresidues, whereas the C-terminal positions of the sets were equimolaramounts of eighteen of the twenty natural (gene-coded) L-amino acidresidues. Binding studies were carried out using those 324 mixtures todetermine which few provided optimal binding to a chosen receptor suchas a monoclonal antibody or live bacterial cells. That study determinedthe two N-terminal optimal binding residues.

Another eighteen sets were then prepared keeping the optimal first twooptimal binding residues, varying the third position among the eighteenL-amino acids used, and keeping the C-terminal three positions asequimolar mixtures. Binding studies were again carried out and anoptimal third position residue was determined. This general procedurewas reported until the entire hexamer sequence was determined.

Similar studies are also reported in Pinilla et al. Vaccines 92, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, pages 25-27 (1992);Appel et al., Immunomethods, 1:17-23 (1992); Houghten et al.,BioTechniques, 13:412-421 (1992); Houghten et al., in Innovation andPerspectives in Solid Phase Syntheses: Peptides. Polypeptides andOligonucleotides, R. Epton (ed.), Intercept, Ltd., Andover, pages237-239 (1992); Houghten et al., in Peptides, J. A. Smith and J. E.Rivier (eds.), Proceedings of the Twelfth American Peptide Symposium,ESCOM, Leiden, pages 560-561 (1992); and WO 92/09300 published Jun. 11,1992.

A still different approach was reported in Pinilla et al.,BioTechniques, 13:901-905 (1992). In that report, a total of 108 freehexamer peptide mixture sets were prepared. Those sets contained one ofeighteen amino acid residues at each of the six positions of the hexamerchains, with the other five positions being occupied by equimolaramounts of those same eighteen residues. Again, over 34 milliondifferent peptides were represented by those 108 sets (6 positions×18residues/position).

Each of the sets was assayed for binding to a monoclonal antibody asreceptor. The residue aposach position that provided best bindingresults for that position provided a peptide sequence that was identicalto the known epitope for that monoclonal. This process also providedsequences for other peptides that were bound almost as well by themonoclonal.

The above work with and implications from use of oligopeptidesnotwithstanding, oligopeptide life times in in vivo systems where thepeptide is introduced by injection or inhalation are typically quiteshort due to hydrolysis and other degradative mechanisms that depend onthe peptide bond. Hydrolysis, both by enzymes and stomach acids, canalso limit peroral administration of otherwise active oligopeptides.

The availability of a wide variety of clearly identified, hydrolyticallystable peptides or peptide-like molecules in relatively limited mixtureswould greatly facilitate the search for optimal molecules for anyparticular therapeutic end use application.

It would therefore be of considerable interest to have a method for thesynthesis of mixtures of peptide-like molecules that are stable toenzymatic hydrolysis and in which individual amino acid residuepositions can be specifically defined, such that a comprehensive arrayof molecules is available to researchers for the identification of oneor more of the optimal molecules for reaction with receptors (acceptors)of interest, from which one can derive optimum therapeutic materials fortreatment of various organism dysfunctions. The disclosure that followsdiscusses one such group of peptide-like molecules that are more stableto enzymatic hydrolysis than are peptides themselves.

BRIEF SUMMARY OF THE INVENTION

One aspect of the invention contemplates a set of linear peralkylatedoligopeptides comprising a mixture of equimolar amounts of linear C₁ -C₇-alkyl peralkylated oligopeptide chain members containing the samenumber of about two to about ten peralkylated amino acid residues ineach oligopeptide chain. Each of the peralkylated amino acid residuesexcept proline has its peptidyl amido nitrogen atom alkylated with a C₁-C₇ -alkyl group. Amino acid side chains and the N-terminal amine group,if present, can also be alkylated so that each alkylated peptide in themixture can be viewed as a peralkylated oligopeptide. Removal of alkylester groups from alkylated carboxylic acid side chains to form carboxylgroups is also contemplated. The members of the set have one or morepredetermined, known peralkylated amino acid residues at the same one ormore predetermined positions of the oligopeptide chain, and the libraryhas equimolar amounts of at least six different peralkylated amino acidresidues at one or more of the same other positions of the peralkylatedoligopeptide chain. The amino-terminus of each oligopeptide is aquaternary C₁ -C₇ -alkylammonium group, an amino group, an N--C₁ -C₇-alkylamino group or an N--C₁ -C₇ -alkyl-N--C₁ -C₁₈ hydrocarboyl or apyroglutamoyl group. The carboxy-terminus is a C₁ -C₇ -alkyl carboxylicacid ester, mono- or di-N--C₁ -C₇ -alkylcarboxamide or a carboxyl group.A C₁ -C₇ -alkyl group at the N- or C-terminus is a C₁ -C₇ -alkyl groupthat can be the same or different from the other C₁ -C₇ -alkyl groupspresent in each molecule.

The one or more peralkylated amino acid residues at the same one or morepredetermined positions of the peralkylated oligopeptide chain arepreferably at a predetermined position that is adjacent to one terminus,and more preferably that one terminus is the amino-terminus. The firsttwo peralkylated amino acid residues at the same one or morepredetermined positions are adjacent to the amino-terminus in anotherpreferred embodiment. The equimolar amounts of peralkylated amino acidresidues are at one or more positions that are adjacent to one terminusin another preferred embodiment, and more preferably, the one terminusis the carboxy-terminus.

A set of peralkylated oligopeptide chains preferably contains five toabout eight peralkylated amino acid residues in each chain. A pluralityof sets of sets (a library) of peralkylated oligopeptides are alsocontemplated. One such library contains sets in which only one positionis occupied by a predetermined peralkylated amino acid residue, whereasthe other positions of each set are occupied by equimolar mixtures ofperalkylated amino acid residues. The number of such sets in a libraryis equal to the number of positions in the oligopeptide chain (thelength of the peralkylated peptide) multiplied by the number ofperalkylated amino acid residues at each position of the chain. Theselibraries are sometimes referred to as positional libraries.

Another aspect of the invention contemplates a process for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor. Such a process comprises the steps of:

(a) providing a library of sets of linear C₁ -C₇ -alkyl peralkylatedoligopeptides in which each set comprises a mixture of equimolar amountsof peralkylated oligopeptide member chains containing the same number oftwo to about ten peralkylated amino acid residues in each peralkylatedoligopeptide chain. The member chains of each set have one or more of atleast six different predetermined peralkylated amino acid residues atone or more predetermined positions of the peralkylated oligopeptidechain, and each set has an equimolar amount of at least six differentperalkylated amino acid residues at the same one or more other positionsof the peralkylated oligopeptide chain. The amino-terminus of each ofthe peralkylated oligopeptides in the set is a quaternary alkylammonium,amino, N-alkylamino, C₁ -C₁₈ hydrocarboylalkylamido or pyroglutamoylgroup, and the carboxy-terminus is an alkylamido, alkylcarboxylate orcarboxyl group. A C₁ -C₇ -alkyl group at the N- or C-terminus is a C₁-C₇ -alkyl group that can be the same or different from the other C₁ -C₇-alkyl groups present in each molecule. The sets of the library differin that the one or more predetermined peralkylated amino acid residuespresent at the one or more predetermined chain positions within each setare different between the sets.

(b) Each set from that library of sets is separately admixed with theacceptor in an aqueous medium at a set concentration of about 0.1milligrams per liter to about 100 grams per liter. The binding of eachset to the acceptor is separately assayed. A set exhibitingpreferential, specific binding relative to the other sets is determined,thereby identifying a peralkylated amino acid residue that providedpreferential binding at said one or more predetermined positions.

(c) A second library of sets of linear peralkylated oligopeptides isprovided in which each set comprises a mixture of equimolar amounts ofC₁ -C₇ -alkyl peralkylated oligopeptide member chains containing thesame number of two to about ten peralkylated amino acid residues in eachchain as the chains of the first-named plurality of sets. The memberchains of each second library of sets contain the one or moreperalkylated amino acid residues of the first-named set identified asexhibiting preferential, specific binding in the same one or morepredetermined chain positions as the first-named sets. The member chainsof the second sets have a predetermined one of the at least sixdifferent peralkylated amino acid residues at another predeterminedposition of the peralkylated oligopeptide chain different from the oneor more positions of the identified peralkylated amino acid residue(s)of the first-named plurality of sets. Each of the second library of sets(a) has equimolar amounts of at least six different peralkylated aminoacid residues of the first-named sets at the same one or more positionsof the peralkylated oligopeptide chain not occupied by the one or moreidentified peralkylated amino acid residues or the predeterminedperalkylated amino acid residues, (b) has one fewer peralkylated aminoacid residue positions occupied by equimolar amounts of at least sixdifferent peralkylated amino acid residues, and (c) has the same firstand second termini as the peralkylated oligopeptides of said first-namedset.

(d) Each set of the second library of sets is separately admixed withthe acceptor in an aqueous medium at a set concentration of about 0.1milligrams per liter to about 100 grams per liter. The binding of eachset to the acceptor is separately assayed. A second set exhibitingpreferential, specific binding is determined, thereby identifying aperalkylated amino acid residue that provides preferential binding atthe other predetermined position in the peralkylated oligopeptide chain;

(e) Steps (c) and (d) are repeated using zero through seven furtherlibraries of sets of peralkylated oligopeptides instead of the secondlibrary of sets. Each further library of sets of peralkylatedoligopeptides comprises a mixture of equimolar amounts of member linearperalkylated oligopeptide chains containing the same number of two toabout ten peralkylated amino acid residues in each peralkylatedoligopeptide chain as the chains of the first-named library of sets. Themember chains of each further library of sets contain the peralkylatedamino acid residues in the oligopeptide chain positions that exhibitedpreferential, specific binding in a library of sets used immediatelybefore and a predetermined one of the at least six differentperalkylated amino acid residues at another predetermined position ofthe alkylated chain different from the positions of the identifiedperalkylated amino acid residues of the library of sets used immediatelybefore. Each of the further library of sets has equimolar amounts of theat least six different peralkylated amino acid residues of thefirst-named sets at the same one or more positions of the peralkylatedoligopeptide chain not occupied by the identified peralkylated aminoacid residues or the predetermined peralkylated amino acid residues, andhas the same first and second termini as the peralkylated oligopeptideof the first-named library set.

(f) At least six peralkylated oligopeptide chains are provided in whicheach chain contains the same number of two to about ten peralkylatedamino acid residues in each peralkylated oligopeptide chain as thechains of the first-named library of sets. Each peralkylatedoligopeptide chain contains the identified peralkylated amino acidresidues in the peralkylated oligopeptide chain positions that exhibitedpreferential, specific binding in step (e), a predetermined one of theat least six different peralkylated amino acid residues at anotherpredetermined position in the peralkylated chain different from thepositions of the identified substituent reduced amino acid side chainsused in step (e), and has the same N- and C-termini as the sets of thefirst-named library of sets.

(g) Each of the at least six peralkylated oligopeptides of (f) isseparately admixed with the acceptor in an aqueous medium at asubstituted peralkylated oligopeptide concentration of about 0.1milligrams to about 100 grams per liter. The binding of eachperalkylated oligopeptide is separately assayed. The peralkylatedoligopeptide exhibiting preferential, specific binding is determined,thereby determining the sequence of a linear peralkylated oligopeptidethat preferentially binds to the acceptor.

The before-discussed preferences as to the sets hold where the sets areused in an above assay. In addition, it is preferred that the identifiedand predetermined substituent peralkylated amino acid residues areadjacent to each other. More preferably, the predetermined one or moreof at least six peralkylated amino acid residues at one or morepredetermined positions of (a) include a terminal residue position ofthe peralkylated oligopeptide chain.

It is also preferred that the first-named peralkylated oligopeptidechains contain about 5 to about 8 peralkylated residues. It is furtherpreferred that at least ten different peralkylated acid residues areutilized instead of at least six.

In one preferred process, the acceptor is a cellular receptor. Morepreferably, the cellular receptor is present in a bacterium or yeastcell cultured in a growth medium.

Yet another embodiment is another process for determining the sequenceof a peralkylated oligopeptide that preferentially binds to an acceptor.This process comprises the steps of:

(a) providing separate libraries or pluralities of sets of linearperalkylated oligopeptides. Each set of those libraries comprises amixture of equimolar amounts of C₁ -C₇ -alkyl peralkylated oligopeptidechains containing the same number of two to about ten peralkylated aminoacid residues in each chain. Each peralkylated oligopeptide chain has asingle one of at least six different predetermined peralkylated aminoacid residues at a single predetermined position of the peralkylatedoligopeptide chain, and each set has equimolar amounts of each of the atleast six different peralkylated amino acid residues at the otherpositions of the peralkylated oligopeptide chain. Each set differs fromthe other sets in the identity and chain position of the singlepredetermined peralkylated amino acid residue present at thepredetermined position within the set. The amino-terminus of each of theperalkylated oligopeptides in the set is a quaternary alkylammoniumgroup, an amino group, an N-alkylamino group, an N-alkyl-N--C₁ -C₁₈hydrocarboyl group or a pyroglutamoyl group, and the carboxy-terminus isa mono- or di-alkylcarboxamide, alkylcarboxylate or carboxyl group. A C₁-C₇ -alkyl group at the N- or C-terminus is a C₁ -C₇ -alkyl group thatcan be the same or different from the other C₁ -C₇ -alkyl groups presentin each molecule.

(b) Each set is separately admixed with the acceptor in an aqueousmedium at a set concentration of about 0.1 milligrams per liter to about100 grams per liter, and the binding of each set to the acceptor isseparately assayed. The peralkylated amino acid residue that exhibitedpreferential, specific binding at each position of the peralkylatedoligopeptide chain provides the sequence of a peralkylated oligopeptidethat preferentially binds to the acceptor, or one or more peralkylatedresidues that are important to that binding.

The before-discussed preferences for the sets also hold here. Inaddition, it is preferred that the single, predetermined positions ofthe library of sets, taken as a group, are adjacent to each other in theperalkylated oligopeptide chain. Each peralkylated chain also preferablycontains about 5 to about 8 peralkylated amino acid residues.

It is also preferred that the single predetermined peralkylated aminoacid residue of each peralkylated oligopeptide chain is one of at leastten different peralkylated amino acid residues, and the same at leastten different peralkylated amino acid residues are present in equimolaramounts at the other peralkylated oligopeptide positions of each set.

Also contemplated is a set of linear C₁ -C₇ -alkyl peralkylatedoligopeptides as described before that is linked to a solid support,such as a solid phase synthesis resin. The peralkylated oligopeptidesare individually linked the solid support by a selectively severablebond such as an amide or ester bond.

Specific permethylated oligopeptides are also contemplated. One group ofpermethylated oligopeptides has the formula

    Xaa.sub.1 PheXaa.sub.3 PheXaa.sub.5 Xaa.sub.6              (SEQ ID NO: 35)

wherein Xaa₁ at the first position is an α-trimethylammonium Leu or Pheresidue;

Xaa₃ at the third position is His or Phe;

Xaa₅ at the fifth position is Ile or Phe; and

Xaa₆ at the sixth position is a C-terminal

N-methylcarboxamido His or Phe, with the proviso that at least one ofXaa₅ or Xaa6 is Phe.

Another contemplated permethylated oligopeptide is a permethylatedα-N-terminal trimethylammonium C-terminal N-methylamidooligophenylalanine having 5 through 8 Phe residues.

The present invention has several benefits and advantages.

One salient benefit is that its use can enable one to find a new classof antimicrobial agent that is unlike any previously studied and againstwhich microbes have no defenses.

Another benefit is that the permethylated oligopeptides prepared byperalkylation of a solid support-linked peptide undergoes substantiallyno racemization during the permethylation step.

An advantage of the invention is that the peralkylated oligopeptides areresistant to cleavage by proteolytic enzymes such as trypsin andchymotrypsin.

Another advantage of the invention is that peralkylated oligopeptidesthat are quite toxic to certain Gram positive bacteria causesubstantially no hemolysis of human blood at antimicrobialconcentrations.

Still further benefits and advantages of the invention will be apparentto those skilled in the art from the discussion that follows.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

Peptides are one of a number of fundamental classes of biologicallyrelevant effector molecules. Acceptor systems for peptides include:antibodies, enzymes, membrane-bound and internal cellular receptors.Biologically important peptides include bradykinin, oxytocin,α-endorphins, insulin, and the like. Drug discovery involving peptidesinvariably requires the synthesis and testing of hundreds to thousandsof analogs of the original biologically active sequences. In order tounderstand a given peptide's structure activity relationships, verylarge numbers of peptide analogs are needed in all of these areas.

The diversity of the combinatorial possibilities of even the 20 naturalamino acids makes usually-used synthesis methods sorely limited in thetask of screening for optimal peptide antigens, peptide ligands forbiologically relevant acceptor systems, enzyme inhibitors,antimicrobials, and the like i.e., there are 64,000,000 possible sixresidue peptides (20⁶), 1,280,000,000 possible seven residue peptides(20⁷), and the like!. Although the usually-used methods for singlepeptide syntheses have greatly facilitated studies with syntheticpeptides, and are available commercially either on a custom basis or foruse in kit form, they permit only a very small fraction of possibleoligopeptides (composed of either natural or unnatural amino acids) tobe prepared.

Equimolar amounts of each component making up the library (or memberset) to be studied could be expected to ensure the necessary selectivityof the interactions of the desired peralkylated oligopeptide in themixture to be used (i.e., the "needle in the haystack"-finding thecorrect peralkylated hexapeptide in the 64,000,000 possible combinationsof the 20 natural amino acid side chains would be analogous to finding asingle steel needle in 63,999,999 copper needles). As an insight intothe extreme selection criterion involved in such a system, it is helpfulif one considers that a single six-letter word would have to be readilyfound in the presence of 63,999,999 other six-letter words (63,999,999six-letter words would fill approximately 50,000 pages of text of thesize found in a usual scientific journal).

The present invention relates to oligomeric peptide-like molecules thathave peralkylated amino acid residues. The peptide-like molecules ofthis invention are C₁ -C₇ -alkyl peralkylated oligopeptides. The presentinvention relates generally to linear peralkylated oligopeptides thatcontain 2 to about 10 peralkylated amino acid residues.

Mixtures of linear C₁ -C₇ -alkyl peralkylated oligopeptides areparticularly contemplated herein, and a mixture that contains one ormore predetermined peralkylated amino acids at one or more predeterminedpositions of the peralkylated oligopeptide chain with the remaining oneor more positions occupied by described equimolar mixtures ofperalkylated residues are referred to as a peralkylated oligopeptideset. Such a mixture or set is preferably prepared by peralkylation of acorresponding mixture of oligopeptides.

A plurality of related peralkylated oligopeptide sets constitute alibrary of sets. The member sets of a library of sets, also referred tosimply as a library, have the same length and termini, and have the samenumber chain positions occupied by equimolar mixtures of the same atleast six different peralkylated amino acid residues. The member setsdiffer from each other in (a) the position of the one or morepredetermined peralkylated amino acid residue, (b) the identity of theone or more predetermined peralkylated amino acid residue, or (c) boththe position and identify of the one or more predetermined peralkylatedamino acid residue.

One exemplary library of 400 sets of hexamers has the N-terminal firsttwo positions occupied by each of the 20 peralkylated naturallyoccurring (gene coded) amino acid residues, and the C-terminal fourresidue positions occupied by equimolar mixtures of at least sixperalkylated naturally occurring amino acid residues. Another library isitself a library of six libraries of hexamers, and contains a total of120 member sets. A first member library contains twenty member sets inwhich position 1 from the N-terminus is occupied by each of theperalkylated twenty naturally occurring amino acids, with the remainingfive positions occupied by mixtures. Another library of twenty membersets has position 2 occupied by each of those peralkylated residues andthe remaining positions occupied by mixtures. Similar twenty-member setlibraries are contemplated in which each of the remaining five positionsis occupied by one of those 20 peralkylated residues and the remainingpositions are equimolar mixtures of those peralkylated residues.

A contemplated C₁ -C₇ -alkyl peralkylated oligopeptide of a set isalkylated at each position of a precursor oligopeptide that bore anactive hydrogen. Consequently, each set is also peralkylated, as is alibrary of sets.

The side chains of many amino acids are unchanged on peralkylation sothat, for example, the methyl side chain of an alanine amino acidresidue in an oligopeptide is a methyl side chain of a peralkylatedoligopeptide. Hydroxyl group-containing side chains are usuallyprotected from alkylation but can be alkylated if desired. Amino acidresidue side chains containing carboxyl, amido, guanidino, mercapto,amino, and aza-substitutions contain active hydrogens, and are not inertto the contemplated, preferred peralkylation process. As such, the sidechains of aspartic and glutamic acids, asparagine and glutamine, and theguanidino group-containing side chain of arginine are not contemplated.

Rather, all atoms with an active hydrogen atom are alkylated. Thus, theN-terminal α-amino group, when present as such, is formed into aquaternary C₁ -C₇ -alkylammonium group, whereas when an N-terminal C₁-C₁₈ acyl (hydrocarboyl) group such as acetyl is present, anN-acyl-N-alkylamino, C₁ -C₁₈ hydrocarboylalkylamido group is formed. AnN-alkyl-N-pyroglutamoyl group is formed where an N-pyroglutamoyl groupwas present. Similarly, each of the amido groups, except that of aproline (prolyl) that forms a secondary amido peptidyl bond, isalkylated to the greatest extent possible so that the terminal amidogroups of Gln and Asn become dialkylated. Side chain blocking groupsused during synthesis are usually removed so that carboxyls of Asp andGlu become alkyl carboxylate esters and Cys forms an alkyl thio ether.The aza nitrogens of His and Trp become N-alkylated and the amino andguanidino groups of Lys and Arg become quaternary, alkylammonium groups.The benzyl protecting groups of Ser and Thr are typically not removed,and Met is preferably used as the sulfoxide, which is also stable toalkylation, and can be maintained in a peralkylated set, or reduced, asdesired.

Where a precursor oligopeptide is synthesized with its C-terminalresidue bonded to a benzhydrylamine (BHA) or methylbenzhydrylamine(MBHA) resin as solid support, the C-terminal residue becomes amono-alkylated carboxamide if alkylation is done prior to cleavage fromthe resin and a di-alkylated amide if alkylation is carried out afterresin cleavage. Where the C-terminal residue is linked to the solidsynthesis support via an ester bond, a C-terminal alkyl carboxylateester is formed. Reaction of a peralkylated oligopeptide set containinga C-terminal ester group with a mono- or di-C₁ -C₇ -alkyl amine formsthe corresponding C-terminal C₁ -C₇ -alkyl carboxamide. Reaction of aperalkylated oligopeptide or peralkylated oligopeptide set that containsone or more alkyl carboxylate ester groups with an aqueous base such assodium hydroxide transforms the ester into a carboxyl group. Thatcarboxyl group can thereafter be re-esterified with the same or adifferent C₁ -C₇ -alkyl group using usual esterification techniques.

It is also noted that treatment of a C₁ -C₇ -alkyl peralkylatedoligopeptide or set of C₁ -C₇ -alkyl peralkylated oligopeptides thatcontain an N-terminal C₁ -C₁₈ acyl group, and particularly an N-benzoylgroup with acid, under acidic conditions causes the amino-terminalresidue to be removed, leaving an N-alkylamine as the amino-terminalgroup of the peralkylated oligopeptide or set that is one residueshorter than its precursor set. Thus, where an N-terminal C₁ -C₁₈ acylgroup is desired in the peralkylated oligopeptide, that group is addedafter cleavage from the solid support.

The contemplated sets of linear peralkylated oligopeptides arepreferably prepared from corresponding sets or mixtures of oligopeptidesby C₁ -C₇ -alkyl peralkylation of the mixture. An individual linearperalkylated oligopeptide is preferably also prepared from acorresponding individual oligopeptide. As a consequence, in thedescription below, the invention will be described in a preferredembodiment in which the linear peralkylated oligopeptides are preparedfrom precursor oligopeptides that do or can contain most or all of thetwenty naturally occurring amino acid residues. It will be understood,however, that the invention can be used with at least six differentamino acid residues, and more than twenty different residues.

For instance, an oligopeptide can include the naturally occurring 20amino acids, one or both isomers of ornithine, norleucine,hydroxyproline, β-alanine and the other C₄ -C₆ amino acids such asγ-aminobutyric and ε-aminocaproic acids and the D-stereoisomers of thenaturally occurring twenty amino acids, so that use of about fiftyprotected D- and L-amino acids is contemplated for synthesis. Precursoroligopeptide sets and oligopeptide mixture pools (discussed hereinafter)that contain all D-amino acid residues and mixtures of both D- andL-forms are contemplated for use in preparing corresponding linearperalkylated oligopeptides and linear peralkylated oligopeptide sets.Consequently, as used herein, the term "amino acid" will, unlessotherwise stated, be intended to include not only the naturallyoccurring (genetically coded) L-amino acids but also theirD-stereoisomers and unnatural amino acids. The phrases "amino acidderivative", "protected amino acid derivative" or the like are usedherein for a protected amino acid added as a reactant, whereas thephrase "amino acid residue", "residue" or the like is used herein for areacted protected amino acid that is a portion of an oligopeptide chain.

Further, the terms "peptide" and "oligopeptide" are considered to besynonymous (as is commonly recognized) and each term can be usedinterchangeably as the context requires. The word "polypeptide" is usedfor chains containing more than ten amino acid residues. Alloligopeptide and polypeptide formulas or sequences shown herein arewritten from left to right and in the direction from amino-terminus tocarboxy-terminus.

The abbreviations used herein for derivatives and residues of the twentynatural amino acids are reproduced in the following Table ofCorrespondence:

    ______________________________________                                        TABLE OF CORRESPONDENCE                                                       Abbreviation                                                                  1-Letter 3-Letter        Amino Acid                                           ______________________________________                                        Y        Tyr             tyrosine                                             G        Gly             glycine                                              F        Phe             phenylalanine                                        M        Met             methionine                                           A        Ala             alanine                                              S        Ser             serine                                               I        Ile             isoleucine                                           L        Leu             leucine                                              T        Thr             threonine                                            V        Val             valine                                               P        Pro             proline                                              K        Lys             lysine                                               H        His             histidine                                            Q        Gln             glutamine                                            E        Glu             glutamic acid                                        W        Trp             tryptophan                                           R        Arg             arginine                                             D        Asp             aspartic acid                                        N        Asn             asparagine                                           C        Cys             cysteine                                             X        Xaa             another residue,                                                              or one of several                                                             residues                                             ______________________________________                                    

The word "predetermined" is used in two contexts herein, and has asimilar meaning in each context.

A "predetermined" amino acid residue is a single residue whose identityis known or specifically defined, e.g., alanine, glycine, tyrosine,etc., as compared to being a mixture of residues. A linear peralkylatedoligopeptide or set thereof similarly contains a before-definedperalkylated amino acid side chain whose identity is known orspecifically defined.

A "predetermined position" in an oligopeptide mixture sequence or chainis a position, from and including the amino-terminal residue as position1, occupied by a predetermined amino acid residue or of a mixture ofresidues, and which position is known and specifically identified. Alinear peralkylated oligopeptide similarly contains a peralkylated aminoacid residue at a particular position in the chain and, a set of suchperalkylated oligopeptides also contains a mixture of peralkylated aminoacid residues at other known or specified position(s) in the chain.

The letter "O" is used herein to indicate a predetermined, butunspecified single amino acid residue or the peralkylated amino acidresidue of a peralkylated oligopeptide. Subscripted letters "O", e.g.,O₁, O₂, O₃ . . . O_(n) etc. indicate a predetermined amino acid residueor peralkylated amino acid residue that is predetermined (specified) andat the same position (1, 2, 3 . . . n) among a set of oligopeptidemixtures, solid support-coupled oligopeptide mixtures, in a peralkylatedoligopeptide or set, that is free or solid support-coupled. Thus, asubscripted letter "O" such as O₁ is used where a particular amino acidresidue or peralkylated amino acid residue is intended such as alanineor leucine, whereas an unsubscripted letter "O" is used to mean thateach of the plurality of residues or peralkylated residues is present ata given position, but that that residue or peralkylated residue is notspecified, but is a single residue. Subscripted numbers need not startat the amino-terminus for any given mixture.

The letter "X" is used to indicate that a position in an oligopeptideset or peralkylated oligopeptide set formula occupied by that letter isan equimolar mixture of each of at least six amino acid residues coupledor peralkylated residues, and preferably ten or more such residues orperalkylated residues, and more preferably about 15 to about 20.

The letter "B" is used to indicate a solid support used in the synthesesdescribed herein, such as a particulate resin.

A peralkylated oligopeptide "corresponds" to a precursor oligopeptidewhen the former is the peralkylated form of the latter, and vice versa.In addition, an illustrated peralkylated oligopeptide sequence will beusually prefaced by the word "PerA" to further distinguish aperalkylated oligopeptide from its corresponding oligopeptide. Theprefix "PerM" is used for a preferred permethylated oligopeptide.

For example, a trimer oligopeptide pool linked to a solid support whosefirst position is defined and whose second and third positions aremixtures can be represented as O₁ XX--B. Similarly, a set of preferredlinear peralkylated oligopeptides having six peralkylated residues whosesecond and third positions contain predetermined peralkylated amino acidresidues, whose remaining positions are occupied by mixtures ofperalkylated residues, whose N-terminal nitrogen atom is bonded to threealkyl groups such as methyl and whose C-terminus is an N-alkyl groupsuch as N-methyl can be depicted as PerA-N(CH₃)₃ --X₁ O₂ O₃XXX--NH(CH₃).

A contemplated linear peralkylated oligopeptide set contains at leastone (one or more) predetermined peralkylated amino acid residues at atleast one (one or more) predetermined oligopeptide chain position andmixtures of at least six peralkylated amino acid residues used forsynthesis at at least one (one or more) other position of theperalkylated oligopeptide chain. At least six different peralkylatedamino acid residues are present at the mixed positions and one of thosesame six peralkylated residues is preferably the at least onepredetermined peralkylated residue for a given set with an exceptiondiscussed hereinafter. In preferred practice at least ten differentperalkylated amino acid residues are used, and more preferably still,about 15 to about 20 peralkylated amino acid residues are used at themixture positions and each can constitute the single, predeterminedperalkylated residue at the at least one predetermined position.

Thus, the peralkylated residue of that at least one predeterminedposition can be one of at least six, preferably at least ten or morepreferably about 15 to about 20 peralkylated residues. That at least oneperalkylated residue is usually referred to herein as "a predeterminedperalkylated amino acid residue", whereas in other instances thatperalkylated residue is described as "one of at least six peralkylatedamino acid residues" or the like.

Synthesis Processes

Overview

The preparation of a set of linear peralkylated oligopeptides having apredetermined C₁ -C₇ -alkyl peralkylated residue at at least oneposition and equimolar amounts of at least six other desiredperalkylated residues at at least one other position preferably beginswith the preparation of a corresponding oligopeptide set that isthereafter peralklyated. Equimolarity being of importance for theperalkylated residues of the mixture positions, synthesis of thecorresponding oligopeptide set is of importance.

Two general approaches to such syntheses are preferred. One is referredto as the physical mixture process and the other is referred to as thechemical mixture process. Both approaches utilize a solid phase supportsuch as a benzyhydrylamine (BHA) or methylbenzhydrylamine (MBHA) resincommonly used in solid phase peptide syntheses, as are discussedhereinbelow.

The physical mixture process utilized is that described in Houghten etal., Letters to Nature, 354:84-86 (1991); Pinnila et al., Vaccines 92,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pages 25-27(1992); Appel et al., Immunomethods, 1:17-23 (1992); and WO 92/09300published Jun. 11, 1992. These synthetic processes are also similar tothe processes disclosed in Furka et al., Int. J. Peptide Protein Res.,37:487-493 (1991), Huebner et al. U.S. Pat. No. 5,182,366, incorporatedby reference, and Lam et al., Letters to Nature, 355:82-84 (1991).

The latter two processes and that used for preparing precursoroligopeptide sets herein differ in concept. In both Lam et al. andHuebner et al., the desired peptide is selected by its binding orreaction, recovered and then its sequence is determined. Furka et al.teach no reactions with their mixtures. The present precursoroligopeptide sets are prepared with one or more known, predeterminedresidues at one or more known, predetermined positions along the chainso that all one need do is determine which oligopeptide of knownsequence bound to the acceptor used. That same concept is used for theultimately produced peralkylated oligopeptides.

A chemical mixture synthesis of a precursor oligopeptide set can be oneof those described in Rutter et al. U.S. Pat. No. 5,010,175 or GeysenU.S. Pat. No. 5,194,392, whose disclosures are incorporated byreference, or as described in the previously noted published papers ofwhich Geysen is an author.

Both Rutter et al. and Geysen report using N-t-BOC protecting groups fortheir chemical mixture syntheses. Each of those patents provides anexemplary mixture of N-t-BOC-blocked amino acid derivatives for use insynthesis of equimolar amounts of amino acid residues.

It is noted that the present invention is not limited to use of N-t-BOCblocking groups for synthesis of precursor oligopeptide sets. This isthe case whether the physical or chemical mixture approaches areutilized. Thus, any blocking group can be utilized. Table 1, below,provides mole ratios of blocked amino acids that can be used for achemical mixture synthesis using Fmoc blocking group chemistry.

                  TABLE 1*                                                        ______________________________________                                        Amino Acid     Mole Ratio                                                     ______________________________________                                        Ala            0.22                                                           ASP(.sup.t BU ester)                                                                         0.47                                                           G1U(.sup.t BU ester)                                                                         0.62                                                           Phe            0.35                                                           Gly            0.20                                                           His (Tr)       0.72                                                           Ile            2.51                                                           Lys (.sup.t Boc)                                                                             0.59                                                           Leu            0.48                                                           Met            0.34                                                           Asn            1.65                                                           Pro            0.20                                                           Gln            2.03                                                           Arg (Mtr)      1.98                                                           Ser(.sup.t Bu ether)                                                                         0.80                                                           Thr(.sup.t Bu ether)                                                                         2.18                                                           Val            1.85                                                           Tyr(.sup.t Bu ether)                                                                         0.81                                                           Trp            0.99                                                           ______________________________________                                         *Parenthesized designations in the left column are protecting groups.         .sup.t Bu = tbutyl; Tr = trityl; .sup.t Boc = tbutyloxycarbonyl; Mtr =        4methoxy-2,3,6-trimethylbenzenesulfonyl.                                 

Substantial equimolarity in the mixture positions is typically withinthe limits of weighing accuracy using the physical mixture syntheticprocess because single amino acids are reacted in large excess andreactions are driven to completion. The chemical mixture process doesnot provide exact equimolarity as does the physical mixture processdescribed before. For example, U.S. Pat. No. 5,010,175 reportedvariation from equimolarity in the range of 0.20-0.32 moles and anaverage of 0.25±0.04, with each amino acid being no more than 0.8 to1.28 times the desired result. Deviations from equimolarity from thatobtained with the physical mixture method of up to about 35 percent havebeen observed with no adverse effect. Regardless of the deviations fromexact equimolarity observed from use of the chemical mixture method, thevarious oligopeptides required to obtain enhanced binding by acorresponding peralkylated oligopeptide are present in large enoughquantities to be useful in the assay methods discussed hereinafter.

It is thus seen that both physical and chemical mixture syntheticprocesses for preparing a desired precursor oligopeptide set are wellknown in the art. In addition, Examples 1, 2 and 3 herein discuss andillustrate exemplary syntheses using both types of syntheses.

It is noted that cysteine and tryptophan are frequently omitted fromprecursor oligopeptide sets and corresponding linear peralkylatedoligopeptide sets because of side reactions that can occur from theiruse. It has been found, however, that use of an N-formyl blocking groupon tryptophan can alleviate much of the difficulty in synthesis whenthat residue is incorporated into an oligopeptide chain.

It is further noted that one can use a wide range of solid supports fora contemplated synthesis of an oligopeptide set. Usually usedcross-linked styrene beads having benzhydrylamine groups are a preferredsolid support. However, many other solid supports as are disclosed inU.S. Pat. No. 4,631,211 can also be utilized, as can a cellulosicsupport such as cotton as is described in Lebl et al. U.S. Pat. No.5,202,418, both of whose disclosures are incorporated by reference.

C. Termini and Coupling

In preferred practice, each oligopeptide is coupled to the solid supportduring synthesis by a selectively severable covalent bond, such as anester or an amide bond. An ultimately produced oligopeptide mixture setcan be cleaved (separated or severed) from the solid support, recoveredand thereafter peralkylated to form a free linear peralkylatedoligopeptide set, or the alkylation can be carried out while theoligopeptide is coupled to the solid support (oligopeptide mixture pool)to form a solid support-coupled linear peralkylated oligopeptide mixtureset.

As noted earlier, each peralkylated oligopeptide contains a chain of twoto about ten peralkylated residues, and more preferably about five toabout eight peralkylated residues so that a precursor oligopeptide setmember contains a chain having two to about ten reacted amino acidresidue repeating units. More preferably, each precursor oligopeptidecontains a chain of about five to about eight reacted amino acidresidues.

A C₁ -C₁₈ straight or branched chain acyl (hydrocarboyl) orpyroglutamoyl group is often bonded to the N-terminus a-amine of anoligopeptide so that after deblocking, alkylation and cleavage from thesolid support, each member chain of a peralkylated oligopeptide setcontains a C₁ -C₁₈ straight or branched chain hydrocarboyl orpyroglutamoyl group. An acetyl group, a C₂ acyl group, is preferred andis often referred to herein as "Ac". Other exemplary C₁ -C₁₈ acyl groupsinclude formyl, propionyl, butyryl, 2-methylpropionyl, hexanoyl,benzoyl, octanoyl, lauroyl, palmitoyl, oleoyl and stearoyl. Hydrogen canalso be present at the amino-terminus α-amine of the precursor chains sothat a quaternary C₁ -C₇ -alkyl ammonium group results at the N-terminusafter peralkylation and cleavage from the solid support.

When the N-terminal α-amine is present as a tri-C₁ -C₇ -alkylammoniumgroup, an anionic counterion is of course present. The counterion chosenis typically univalent to avoid cross-linking and precipitation of theperalkylated oligopeptide. Where a halide alkylating agent is used suchas methyl iodide, benzyl bromide or allyl bromide, that halide is theinitial counterion for the formed tri-C₁ -C₇ -alkyl ammonium group.Cleavage of the peralkylated peptide from the solid support with HF canreplace the initial counterion with a fluoride ion. Any pharmaceuticallyacceptable counterion can be used such as a halide; i.e., fluoride,chloride, bromide or iodide, nitrate or a C₁ -C₆ carboxylate such asformate, acetate, trifluoroacetate, propional, iso-butyrate, hexanoateor the like. A single, desired counterion species can be provided bypassage of the deblocked, severed, peralkylated oligopeptide set throughan anion exchange resin having the desired counterion as theexchangeable anion.

A C₁ -C₁₈ acyl or pyroglutamoyl group is added by reaction of acorresponding anhydride such as acetic anhydride, acid halide such asoctanoyl chloride, by reaction of a suitable activated ester such asN-hydroxysuccinimidyl benzoate or using the free carboxyl group and acarbodiimide such as DCC. An acyl group is usually added to a solidsupport-coupled oligopeptide upon removal of the selectively removableblocking (protecting) group, e.g. N-t-BOC or N-Fmoc, from the N-terminala-amino group.

In those embodiments in which it is desired to have a free N-terminala-amino group on each member of a peralkylated oligopeptide mixture set,a trityl (triphenylmethyl) group is preferably used to block theN-terminal amine prior to the peralkylation step. Once peralkylation iscompleted, the trityl group is removed by standard techniques such astreatment with an acid such as trifluoroacetic acid (TFA; 2 percent) indichloromethane (DCM; 98 percent).

Where a single C₁ -C₇ -alkyl group is desired on the N-terminal amine, asolid support-linked free N-terminal amine-containing peralkylatedoligopeptide mixture pool, prepared using a trityl group as above, isoften first reacted with an excess, e.g., about two-fold, of4,4'-dimethoxydiphenyl methylchloride (DodCl) following the proceduresof Kaljuste et al., Int. J. Peptide Protein Res., 42:118-124 (1993) toform a Dod-protected N-terminal amine. A C₁ -C₇ -alkyl aldehyde orketone can then be used to reductively alkylate the Dod-protectedN-terminal amine using a reducing agent such as sodium cyanoborohydride.The Dod-protecting group is thereafter removed as with 55 percent TFA indichloromethane.

A Dod-protected N-terminal amine is used where monoalkylation is desiredand the alkylating aldehyde or ketone is relatively small and unhinderedas where formaldehyde or acetaldehyde is used. Monoalkylation usingbulkier alkylating agents such as benzaldehyde does not require use of aDod-protecting group, and is accomplished by direct reductive alkylationof the free N-terminal α-amine. Reductive alkylation of the freeN-terminal α-amine with small, unhindered alkylating agents providesN,N-dialkylation. Alkylations between the two extremes can be achievedwith a minimal amount of experimentation as to reaction conditions andreagents that is well within the level of ordinary skill in this art.

Exemplary C₁ -C₇ aldehydes and ketones that can be utilized in thisreductive alkylation include formaldehyde, acetaldehyde, acetone,butanal, methyl ethyl ketone, methyl butyl ketone and benzaldehyde. TheC₁ -C₇ aldehyde or ketone utilized in this step need not form the sameC₁ -C₇ -alkyl group as resulted from the peralkylation step. Preferably,the single N-terminal alkyl group and that present after peralkylationare the same.

Where an oligopeptide mixture pool is coupled to the solid support by anester group formed from the C-terminal residue via a direct bond or anintermediary linker such as a PAM group, and a C-terminal N-alkyl amideis desired in the peralkylated oligopeptide set, the oligopeptide setcan be severed from the solid support by aminolysis using ammonia, andthe resulting C-terminal amide becomes dialkylated when the peptide isalkylated after cleavage. Normal cleavage of an ester group-bondedoligopeptide from the solid support using HF results in a C-terminalcarboxyl group. However, such esters cleave during sodium hydridetreatment resulting in the carboxyl group being alkylated during theperalkylation step. The resulting ester can be cleaved with a base suchas sodium hydroxide to form the corresponding carboxylate group that canbe neutralized to form a free carboxyl group. That carboxyl group canalso be alkylated by standard procedures to form a C₁ -C₇ -alkyl esterwhose alkyl group is different from that of the peralkylatedoligopeptide member chains. Cleavage of an amide-bonded oligopeptidefrom a benzhydrylamine resin solid support with HF prior toperalkylation results in the formation of a C-terminal N,N-dialkyl amidegroup after alkylation, whereas cleavage after peralkylation results information of a C-terminal N-alkyl amide group.

Syntheses of precursor oligopeptide sets is preferably carried out usingforaminous (porous) containers that are described in U.S. Pat. No.4,631,211, whose disclosures are incorporated by reference. Anotheruseful synthetic technique, particularly for use in the chemical mixtureprocess, is the process described in Lebl et al. U.S. Pat. No.5,202,418, whose disclosures are incorporated herein by reference.

Various useful solid supports, methods of their use, reagents forlinking the growing oligopeptide to the support, cleaving anoligopeptide from the support and the like are well known to workersskilled in this art such that further exemplification is unnecessary.Further such exemplifications can, however, be found in U.S. Pat. No.4,631,211 and in WO 92/09300, published Jun. 11, 1992, whose disclosuresare incorporated by reference.

D. Oligopeptide Peralkylation

A contemplated peralkylated oligopeptide set is alkylated at each amidonitrogen of the peptide backbone and side chain group at which an activehydrogen atom is present. An oligopeptide set is peralkylated with areagent that provides a C₁ -C₇ -alkyl group. A precursor oligopeptideset is preferably peralkylated after preparation. A peralkylatedoligopeptide set can also be prepared using appropriate peralkylatedamino acid derivatives, but such a process is not convenient.

Contemplated C₁ -C₇ -alkyl groups with which an oligopeptide set isalkylated include hydrocarbyl and substituted hydrocarbyl alkyl groups.Exemplary hydrocarbyl groups include methyl, which is preferred, ethyl,propyl, iso-propyl, sec-butyl, cyclopentyl, hexyl, heptyl and benzyl,which can also be viewed as a phenyl-substituted methyl group or aralkylgroup.

Substituted C₁ -C₇ -alkyl groups include alkyl carboxamide whose amidonitrogen atoms are themselves substituted with zero, one or two of thesame C₁ -C₃ hydrocarbyl alkyl groups, alkyl hydroxyl and alkylcarboxylate groups. Exemplary of such substituted alkyl groups aremethylcarboxamide --CH₂ C(O)NH₂ !, 2-hydroxyethyl --CH₂ CH₂ OH!,2-hydroxypropyl --C₂ CH(OH)CH₃ ! and 3-carboxypropyl --CH₂ CH₂ CH₂ CO₂ ⁻!. As noted above, a benzyl group can also be viewed as a phenylmethylgroup and can thus be viewed as a substituted alkyl group. The hydroxylsubstituent is typically blocked with a trialkylsilyl group such astrimethylsilyl that can be removed after alkylation withtetrabutylammonium fluoride. A carboxyl-substituted alkyl group ispreferably reacted as its alkali metal salt such as the sodium orpotassium salt.

The peralkylation reaction can be carried out in a number of reactionconditions of non-nucleophilic strong base, solvent, temperature andconcentrations of base and alkylating agent. The peralkylation reactionis a nucleophilic reaction so a polar, aprotic solvent such as dimethylsulfoxide (DMSO), dimethyl formamide (DMF) or hexamethylphosphoramide(HMPA) is preferably used, DMSO being preferred. Minor amounts of theother aprotic solvents such as dioxane and tetrahydrofuran (THF) canalso be present. The peralkylating agent is a before-describedsubstituted or unsubstituted C₁ -C₇ -alkyl group that also includes anappropriate leaving group that is displaced during the reaction such aschloride, bromide, iodide, methylsulfonate or trifluoromethylsulfonate.

A ratio of about 50 to about 500 ml of DMSO or other solvent per gram ofpeptide-linked resin is preferably utilized. More preferably, this rangeis about 100 to about 250 ml/g, with the preferred solvent DMSO.

The alkylating agent is typically used in a large molar excess over themoles of active hydrogens present in the peptide to help assureperalkylation. The alkylating agent is typically used in about 10- toabout 100-fold molar excess over the moles of peptide active hydrogen(potential alkylation sites), and preferably at about a 20- to about80-fold, and more preferably, about 25- to about 40-fold, molar excess.

The non-nucleophilic strong base is preferably an alkali metal hydridesuch as sodium or potassium hydride. More preferably, the milder butstill strong non-nucleophilic base lithium t-butoxide is used. The basemust be of sufficient base strength to remove an active hydrogen from apeptidyl amide bond. An alkali metal hydride or t-butoxide ion has thatrequired base strength and use of either minimizes side productformation as compared with other strong bases.

The non-nucleophilic strong base is used in large excess over the molesof possible peptide alkylation sites (active hydrogens). An about 5- toabout 100-fold excess of base is typically used with an about 10- toabout 30-fold excess being preferred. One preferred non-nucleophilicstrong base is sodium hydride that is preferably used at about 0.25molar in DMSO. The more preferred lithium t-butoxide in THF is used atabout 0.5 molar.

In usual practice, the solid support-coupled precursor peptide issuspended in the solvent and admixed with the non-nucleophilic base fora time period of about 4 to about 24 hours prior to addition of thealkylating agent. A time period of about 16 to about 18 hours at ambientroom temperature is typically used. This time period can be shortened toabout 15-30 minutes with lithium t-butoxide as base.

The peralkylation reaction can be run at temperatures of below zerodegrees C., such as at the temperature of a dry ice/acetone bath, toabout the solvent boiling point, with lower temperatures requiring moretime than higher temperatures. Peralkylation at ambient room temperaturehas been found to be quite effective and convenient and is thuspreferred. The reaction is carried out under anhydrous conditions andpreferably in an inert atmosphere such as under nitrogen or argon; i.e.,in the absence of oxygen.

Methylation of peptide backbone nitrogen atoms has been shown to occurwithin about one minute, with N-terminal amines taking longer topermethylate. Thus, methylation reaction time is another way to preparepermethylated oligopeptide mixture sets having other than quaternaryammonium N-terminal groups. Mono- and dialkylamino N-terminal residueshave been observed at a level that is generally less than about 15percent.

As noted elsewhere, it is preferred to carry out the peralkylation whilethe oligopeptide set is linked to the solid support. Using the peptidesynthesis techniques disclosed in U.S. Pat. No. 4,631,211 that utilizedforaminous containers for the syntheses, several solid support-linkedoligopeptide sets can be peralkylated at one time, each within its ownforaminous container. It is believed that the present invention embodiesthe first report of permethylation of a peptide while that peptide isbonded to a solid support.

For peralkylation carried out with sodium hydride, the base, alkylatingagent and peptide are typically present together in the reactionmixture. Using lithium t-butoxide in THF, the base and peptide areadmixed and reacted, and then substantially all of thet-butoxide-containing liquid phase (as much as possible, e.g. about75-95 percent) is removed. The alkylating agent in DMSO is thereafteradmixed and reacted.

Once the peralkylation is complete, the excess non-nucleophilic strongbase is washed away and neutralized by washing with DMSO, followed byDMF and dichloromethane, and the support-linked peralkylated peptide set(mixture pool) is then dried. Another washing protocol uses three washeswith DMF, two with isopropyl alcohol (IPA), three with DCM and one withmethanol (MeOH) prior to drying. The peralkylated oligopeptide sets canthen be individually cleaved from the solid supports to provide freeperalkylated oligopeptide sets, if desired, or a solid support-linked(-coupled) peralkylated oligopeptide set can be used without cleavagefrom the support.

Alkylation reactions involve the addition of a substituted orunsubstituted alkyl group to an atom such as carbon, oxygen, nitrogen orsulfur. Carbon alkylations are not contemplated here. As contemplatedhere, oxygen, nitrogen or sulfur atoms bonded to active hydrogen atoms;i.e., hydrogen atoms sufficiently acidic to be removed with a strong,non-nucleophilic base, are completely alkylated.

Thus, each peptide bond amide group is alkylated. The N-terminal a-aminogroup is quaternized if free, mono-alkylated if present as an acyl amideor a Dod derivative, or unreacted if blocked as a cyclic imide such assuccinimide, maleimide or phthalimide that is subsequently reacted withhydrazine or blocked with a trityl group.

Alkylation of amino acid residue side chains is a function of theblocking groups used during synthesis of the precursor oligopeptidechains. Thus, the non-reactive side chains that do not use blockinggroups such as those of glycine, alanine, isoleucine, leucine,phenylalanine and proline are not alkylated. The amido side chains ofasparagine and glutamine normally do not require blocking groups, butcontain two active hydrogens on each side chain amide and are eachdialkylated.

Using the usually used blocking groups in a N-t-BOC synthesis asexemplary, the following outlines the results of a contemplatedperalkylation on various amino acid side chains.

Benzyl ether protecting groups of serine and threonine are stable toalkylation as is methionine sulfoxide so that those side chains are notalkylated where alkylation is carried out prior to deblocking.Methionine sulfoxide can be used as is, or reduced for use.

The formyl group preferably used to block the aza nitrogen of tryptophanis lost during alkylation so that an N-alkyl tryptophan (N-alkyl indolering) is formed. The dinitrophenyl blocking group of the one blocked azanitrogen of histidine is lost so that an N-alkyl histidine(N-alkylimidazole ring) results. The usually used β- and γ-benzyl esterblocking groups of aspartic and glutamic acids are lost duringalkylation so those amino acid residues become alkylcarboxylate esters.

The usually used p-toluenesulfonyl, o-chlorobenzyloxycarbonyl andm-bromobenzyloxycarbonyl blocking groups of arginine, lysine andtyrosine are lost during peralkylation. The arginine and lysine residueside chains become quaternary alkylammonium groups, whereas the tyrosinehydroxyl becomes O-alkylated. After initial formation of a lysinequaternary ammonium group, some degradation can occur. Use of a cyclicimido blocking group on the ε-amino group of lysine followed by reactionwith hydrazine can provide a free, unalkylated amine as such an imidonitrogen atom contains no active hydrogens.

Where cysteine is used, which is not preferred, the usually usedp-methoxybenzyl blocking group is partly removed, leading to a mixtureof alkylated and unalkylated mercaptyl groups after deblocking.

It is also noted that a contemplated set can include residues that arenot peralkylated. Such residues can be added after a support-coupledmixture pool of peptides except that N-terminal residue is peralkylatedusing known peptide synthesis techniques for adding individual residuesor mixtures of residues. The non-peralkylated residue is at one terminusor the other, depending upon the direction of peptide synthesis.Inasmuch as C-terminal to N-terminal synthesis is preferred, thenon-peralkylated residues are preferably occupy the N-terminalpositions.

The use of up to six non-peralkylated residues is contemplated. A totalpeptide length of two (zero non-peralkylated residues) to sixteenresidues is contemplated. Preferably, only the N-terminal residue is notperalkylated.

Peralkylated Oligopeptide Sets and Libraries of Sets

One aspect of the present invention contemplates a set of linearperalkylated oligopeptide chains that comprises a mixture of equimolaramounts of linear peralkylated oligopeptide chain members, each memberhaving an amino- and a carboxy-terminus, and containing the same numberof two to about ten peralkylated amino acid residues in each chain. Themembers of the set have one or more (at least one) predeterminedperalkylated amino acid residues at the same one or more predeterminedpositions of the peralkylated oligopeptide chain. A set also hasequimolar amounts of peralkylated amino acid that contain at least sixdifferent peralkylated amino acid residues, preferably including theperalkylated residue at the at least one predetermined position, at oneor more (at least one) of the same other positions of the peralkylatedoligopeptide chain. The amino-terminus of each of the peralkylatedoligopeptides in the set is quaternary alkylammonium, amino,N-alkylamino or a C₁ -C₁₈ hydrocarboylalkylamido group, and thecarboxy-terminus is a mono- or di-alkylamido, alkylcarboxylate orcarboxyl group. A C₁ -C₇ -alkyl group at the N- or C-terminus is a C₁-C₇ -alkyl group that can be the same or different from the other C₁ -C₇-alkyl groups present in each molecule.

A free peralkylated oligopeptide set is preferably prepared from acorresponding set of oligopeptides. A contemplated peralkylatedoligopeptide set can also be solid support-bound and can be preparedfrom a complex mixture pool of solid support-coupled oligopeptides. Forthat reason, exemplary predecessor oligopeptide sets will first bediscussed, with that discussion being followed by a discussion of thecorresponding peralkylated oligopeptide sets, using the support-severed(free) oligopeptide sets and peralkylated oligopeptide sets asillustrative.

A complex mixture pool of solid support-coupled precursor oligopeptides,once deprotected and cleaved or severed from the solid support, isreferred to herein as an oligopeptide set, an oligopeptide mixture set,by a similar phrase, or more simply as a "set". Being severed from thesolid support, a precursor oligopeptide set is unsupported, and becauseof its method of synthesis, such a set is linear.

A precursor oligopeptide mixture set comprises a mixture of equimolaramounts of oligopeptide chains that contain the same number of aminoacid residues in each chain; i.e., have the same chain length of 2 toabout 10 residues, and more preferably about 5 to about 8 residues. Acorresponding linear peralkylated oligopeptide set is similarly amixture of peralkylated peptides of the same length, having the samenumber of 2 to about 10 peralkylated residues, and preferably 5 to about8 peralkylated residues. The amino-terminus of each of the peralkylatedoligopeptides in the set is quaternary alkylammonium, amino,N-alkylamino, a C₁ -C₁₈ hydrocarboylalkylamido or a pyroglutamoyl group,and the carboxy-terminus is a mono- or di-alkylamido, alkylcarboxylateor a carboxyl group. A C₁ -C₇ -alkyl group at the N- or C-terminus is aC₁ -C₇ -alkyl group that can be the same or different from the other C₁-C₇ -alkyl groups present in each molecule.

A precursor oligopeptide set has one or more (at least one)predetermined (specifically defined) amino acid residues at the same oneor more (at least one) predetermined (specifically defined) positions ofthe oligopeptide chain and equimolar amounts of at least six differentamino acid residues, more preferably at least ten different residues,and most preferably about 15 to about 20 different amino acid residues,at one or more (at least one) predetermined (specifically defined) otherpositions of the chain, the one or more predetermined residuespreferably being one of the at least six different residues present inequimolar amounts. When more than one predetermined amino acid residueis present at more than one predetermined position of the chain, thoseresidues can be the same of different. A corresponding peralkylatedoligopeptide set has one or more (at least one) predeterminedperalkylated amino acid residue at the same one or more (at least one)predetermined position of the peralkylated oligopeptide chain, andequimolar amounts of at least six different peralkylated amino acidresidues at one or more (at least one) predetermined positions of thechain.

The number of amino acid residues for the equimolar mixture positions,and thus the number of different sets, is at least six, and morepreferably about ten. Most preferably, that number is about 15 to about20. The same is the case for peralkylated amino acid residues in aperalkylated set. It is often preferred to use 18 (t-BOC-synthesized) or19 (Fmoc-synthesized) sets for each library; i.e., the naturallyoccurring 20 amino acids are used except cysteine that tends tocross-link and tryptophan that is difficult to couple and can alsocross-link. However, tryptophan is often used at a predeterminedterminal position as the at least one predetermined amino acid residueof a set even though it is not one of the residues utilized at equimolarmixtures positions.

In addition, where it is desired to use tryptophan in a precursoroligopeptide set or library, the tryptophan can be added to a growingsolid phase-linked oligopeptide by the use of N.sub.α -t-BOC-N-formyltryptophan that is available from Bachem, Inc., Torrence, Calif. Use ofthe formyl group protects against the adverse side reactions discussedbefore.

Where alkylation is carried out after cleavage from the solid-support,the N-formyl group can be removed during the usual side chaindeprotecting step by the addition of a mercaptan-containing reagent suchas ethanedithiol during the "low HF" deprotection reaction discussedherein. The N-formyl group can also be maintained during the side chaindeprotection step by omission of the mercaptan-containing reagent duringthat step in which case that N-formyl group is replaced by an alkylgroup on alkylation. Where a precursor oligopeptide set is alkylatedwhile still bound to the solid support, the N-formyl group is replacedand the aza nitrogen atom of the indole ring is alkylated.

A preferred precursor oligopeptide mixture set contains the one or morepredetermined residues at one or more predetermined positions thatinclude a chain terminus, most preferably the N-terminus. Such a setalso includes an equimolar amount of at least six different amino acidresidues at one or more predetermined chain positions, and morepreferably those chain positions are adjacent to one another. Inparticularly preferred practice, those adjacent equimolar mixturepositions are at a terminus of the oligopeptide chain, and mostpreferably, that terminus is the C-terminus. Preferably, the samemixture of residues is present at each predetermined position.

In other embodiments, the N-terminal two precursor residues arepredetermined residues within the set, the N-terminal three residues arepredetermined, or the N-terminal four residues are predetermined when aset is six residues long or longer with the other positions beingoccupied by equimolar mixtures of residues. Thus, one or morepredetermined chain positions at the precursor N-terminus are occupiedby predetermined residues and one or more chain positions at theC-terminus are occupied by an equimolar mixture of residues.

In a corresponding linear peralkylated oligopeptide set, it is preferredthat the one or more (at least one) peralkylated amino acid residue is(are) at the N-terminus, with the equimolar peralkylated residue mixturepositions preferably including the C-terminus. It is also preferred thatequimolar peralkylated residue positions be adjacent to each other.

For a precursor set six residues long or longer, an exemplaryoligopeptide mixture set contains equimolar amounts of at least sixdifferent amino acid residues at the carboxy-terminal 1, 2, 3, 4 or 5positions of the oligopeptide chain (i.e., positions 2, 3, 4, 5 and 6from the amino-terminus of a 6-mer), as specifically definedposition(s). At least one other position and preferably more than oneother position of the chain of such a precursor oligopeptide mixture setis occupied by a predetermined amino acid residue whose identity is thesame at an analogous position within the chain for each set, and thosepredetermined amino acid residues are most preferably at anamino-terminal position of the chain, including the amino-terminus ofthe chain. It is to be understood that although the identity of eachpredetermined residue at a given position in the chain is the samewithin each set, each such chain position can be occupied by the same ora different residue as between sets.

Exemplary precursor oligopeptide mixture sets include a dipeptide havingone position predetermined and the other a mixture; a tripeptide havingtwo positions occupied by predetermined residues and the other amixture, or vice versa; a tetrapeptide having one predeterminedposition, e.g. position 1, and three mixture positions; a 5-mer whosefirst position is defined (predetermined) and whose remaining positionsare occupied by mixtures; a 5-mer whose first and fifth positions aredefined and whose second, third and fourth positions are occupied bymixtures; a 6-mer whose first two positions are predetermined and whoselast four are occupied by mixtures; a hexamer whose first threepositions are predetermined and whose last three are occupied bymixtures; a 7-mer whose first position and positions 4-7 are mixturesand whose second and third positions are predetermined; a 7-mer whosefirst, third and fourth positions are predetermined and whose remainingpositions are mixtures; an 8-mer whose third and fourth positions arepredetermined and whose remaining positions are occupied by mixtures ofresidues; an 8-mer whose first four positions are predetermined andwhose last four positions are each mixtures; a 9-mer whose fourth andfifth positions are predetermined, and whose remaining positions aremixtures; a 10-mer whose positions 3-7 are predetermined, and whoseremaining positions are occupied by mixtures; a 10-mer whose firstposition is predetermined, with the remaining positions occupied bymixtures; a 10-mer whose positions 7-9 are predetermined, with theremaining positions occupied by mixtures and the like where each mixtureis an equimolar mixture of a plurality of coupled amino acid residuesthat includes at least 6, and more preferably at least about 10, andmost preferably about 15 to about 20, different amino acid residues asdiscussed previously.

Corresponding linear peralkylated oligopeptide sets are contemplated foreach of the above sets.

Precursor oligopeptide mixture sets that contain two chain positions ofpredetermined amino acid residues and four or more positions ofequimolar mixtures along the chain are among those preferred. For6-mers, those sets have the configurations of predetermined, singleamino acid and equimolar mixtures shown below:

    ______________________________________                                        Predetermined Positions                                                                        Mixture Positions                                            ______________________________________                                        1,2              3-6                                                          2,3              1,4-6                                                        3,4              1,2,5,6                                                      4,5              1-3,6                                                        5,6              1-4                                                          1,3              2,4-6                                                        1,4              2,3,5,6                                                      1,5              2-4,6                                                        1,6              2-5                                                          2,4              1,3,5,6                                                      2,5              1,3,4,6                                                      2,6              1,3-5                                                        3,5              1,2,4,6                                                      3,6              1,2,4,5                                                      4,6              1-3,5                                                        ______________________________________                                    

Each of those positional configurations defines 400 mixture sets whenthe twenty natural amino acids are used. It is preferred that thepredetermined residues, O, be adjacent to each other in the chain.

Precursor oligopeptide mixture sets containing three predeterminedpositions along the chain and three or more equimolar mixture positionsare also preferred. Six-mer sets for those preferred sets have theconfigurations of predetermined, single amino acid and mixtures shownbelow:

    ______________________________________                                        Predetermined Positions                                                                        Mixture Positions                                            ______________________________________                                        1-3              4-6                                                          2-4              1,5,6                                                        3-5              1,2,6                                                        4-6              1-3                                                          1,2,4            3,5,6                                                        1,2,5            2,3,6                                                        1,2,6            3-5                                                          1,3,4            2,5,6                                                        1,4,5            2,3,6                                                        1,5,6            2-3                                                          1,3,5            2,4,6                                                        1,3,6            2,4,5                                                        2,3,5            1,4,6                                                        2,3,6            1,4,5                                                        3,5,6            1,2,4                                                        ______________________________________                                    

Each of the above positional configurations defines 8000 oligopeptidemixture sets when the twenty natural amino acid residues occupy apredetermined position in the chain. It is preferred that the threepredetermined positions be adjacent in the chain.

Using the twenty natural amino acids as exemplary, a precursor 6-mer(hexapeptide) mixture set having only the first position occupied by apredetermined residue has twenty member sets each of which contains 3.2million member oligopeptides. A precursor set having the first twopositions occupied by predetermined residues includes 400 member setseach of which includes 160,000 member oligopeptides.

The discussion as to precursor oligopeptide sets should be taken toapply to corresponding sets of linear peralkylated oligopeptides,including the above-discussed preferences as they apply to correspondingperalkylated sets.

In another particularly preferred embodiment, each precursor setcomprises equimolar amounts of linear oligopeptide chains containing thesame number of two to about ten amino acid residues in each chain. Eachset, and its members, have only one, single, predetermined amino acidresidue e.g. Ala, D-Val, Ser, etc., at a singly predetermined positionof the oligopeptide chain, e.g. positions 1, 2, 3 . . . 10 from theamino-terminus.

Thus, each of the plurality of precursor sets has equimolar amounts ofthe same at least six different amino acid residues at the positionsother than that of the single, predetermined amino acid present at thepredetermined chain position, and that single residue is preferably oneof the same at least six different amino acid residues. Each of theplurality of sets differs from the other sets by the single,predetermined amino acid at the predetermined chain position.

Using a 6-mer corresponding oligopeptide as exemplary, the positions ofpredetermined, single residue and positions of equimolar mixtures ofresidues are shown below.

    ______________________________________                                        Predetermined Positions                                                                        Mixture Positions                                            ______________________________________                                        1                2, 3, 4, 5, 6                                                2                1, 3, 4, 5, 6                                                3                1, 2, 4, 5, 6                                                4                1, 2, 3, 5, 6                                                5                1, 2, 3, 4, 6                                                6                1, 2, 3, 4, 5                                                ______________________________________                                    

There is thus one set of precursor peptides for each of the single,predetermined amino acid residue at position 1. Because at least sixamino acid residues are used in the mixture positions and each of thoseis also preferably used at position 1, the number of the plurality ofposition-1 sets is six. The same is true for each of the otherpositions. The sets defined by the position of the single, predeterminedamino acid residues can be referred to as positional sets.

These positional sets of 6-mers can also be referred to as 5× setsbecause of their five mixture positions. Where the peptides are fiveresidues long or have four mixture positions, the sets can be referredto as 4× sets, and so on.

Because there are six positions in the 6-mer, the number of libraries ofprecursor sets for the above group of positional sets is 6 times 6 or36. There are, however, 6⁶ or 46,656 total oligopeptides represented bythat library of sets. Use of 20 amino acid residues for the mixturepositions of a 6-mer provides 6 times 20 or 120 positional sets, and atotal of 64,000,000 individual oligopeptides.

The single, predetermined amino acid at the predetermined chain positionis utilized in the equimolar mixture of amino acid residues present atthose other positions. If that single, predetermined residue is notpresent in the mixture positions, the binding assay results of a libraryof peralkylated oligopeptides as to that residue lose some meaning as tothat residue.

Corresponding linear peralkylated oligopeptide positional sets have asingle predetermined peralkylated amino acid residue at a singlepredetermined repeating unit position with equimolar amounts of at leastsix different peralkylated amino acid residues at the other repeatingunit positions of the chain. Each of the peralkylated sets differs fromthe other peralkylated sets by the predetermined peralkylated amino acidresidue at the predetermined chain position.

It should be apparent from the foregoing discussion that a plurality ofsets of linear peralkylated oligopeptide sets is also contemplated. Suchpluralities of sets are referred to as libraries of sets. Eachperalkylated oligopeptide member of each set of the library has one ormore predetermined peralkylated amino acid residues at one or morepredetermined positions of the peralkylated oligopeptide chain, the sametermini and the same sequence of equimolar amounts of at least sixdifferent peralkylated amino acid residues at one or more predeterminedpositions in the peralkylated oligopeptide chain. The sets ofperalkylated oligopeptide sets of the library differ in that at leastone predetermined peralkylated residue present at a predeterminedposition within each set is different between the sets.

Exemplary libraries of sets are those corresponding to the previouslydiscussed 400 oligopeptide sets whose first two peralkylatedoligopeptide positions are each occupied by one of the twenty naturallyoccurring peralkylated amino acid residues, and the remaining positions3-6 are occupied by equimolar mixtures. Each member of those 400libraries has two predetermined peralkylated amino acid side chains (O₁and O₂) at one or more predetermined positions (e.g., the N-terminalfirst two positions) and equimolar amounts of the at least six differentperalkylated residues at one or more predetermined positions (e.g., thefour C-terminal positions).

Another exemplary 6-mer set of peralkylated oligopeptide sets has theN-terminal first two positions occupied by predetermined peralkylatedresidues and the remaining positions occupied by mixtures ofperalkylated residues. Similar sets of sets have positions 1-3 occupiedby specific, predetermined peralkylated residues, the fourth positionoccupied by one of the peralkylated amino acid residues used in thestudy, and positions 5 and 6 occupied by mixtures of peralkylatedresidues. Another set of sets has the first four positions defined, thefifth occupied by each of the peralkylated amino acid residues used, andthe sixth position a mixture.

Thus, the above library of sets is comprised of member sets each ofwhich is comprised of a mixture of equimolar amounts of linearperalkylated oligopeptide chains containing the same number ofperalkylated residues in each peralkylated oligopeptide chain; i.e.,here each set has a sequence length of six repeating units. The membersof each set have one to four N-terminal positions occupied by the same,single, predetermined peralkylated amino acid residue (the O₁, O₂, O₃etc. positions) and four to one respective C-terminal positions occupiedby equimolar amounts of at least six different peralkylated amino acidresidues utilized (the equimolar mixture positions, X). The singleposition remaining in each set is the position between those enumeratedabove, and is occupied by one each of the peralkylated amino acidresidues utilized at that position.

The number of sets within a library of sets is determined by the numberof different peralkylated amino acid residues utilized at the above,single remaining position. Thus, where the peralkylated twenty naturallyoccurring amino acid residues are used, each set contains 20 mixtures.The number of individual peralkylated oligopeptides in each mixture of aset is determined by multiplying the numbers of peralkylated amino acidresidues used at each equimolar mixture position.

The linear peralkylated oligopeptide positional sets of exemplary 6-mersets (obtained by alkylation of the previously discussed corresponding120 6-mer oligopeptide sets) each of which contains one predeterminedposition and five mixture positions are also contemplated, andillustrate particularly preferred library of peralkylated oligopeptidesets. Here, again, each set contains a sequence length of six repeatingunits. One position in each set is occupied by one of at least six ofthe predetermined peralkylated amino acid residues utilized for thatposition. The remaining five positions of each set are occupied by equalmolar amounts of at least six different peralkylated amino acidresidues. Again, the number of members of each set is determined by thenumber of predetermined peralkylated residues utilized, and the numberof peralkylated oligopeptides in each set is determined by multiplyingthe numbers of peralkylated residues utilized at each equimolar mixtureposition.

The previously discussed mixtures having equimolar amounts of at leastsix different peralkylated amino acid residues occupying the fourC-terminal positions also constitute a library of sets. Here, the setscontain a sequence length of five to ten peralkylated residues. TheN-terminal peralkylated residue in each set is occupied by each one ofthe predetermined peralkylated amino acid residues utilized at thatposition (O). The repeating unit sequence between the enumeratedN-terminus and four C-terminal positions is the same in each libraryfrom a C-terminal direction to an N-terminal direction.

Still further sets of peralkylated oligopeptides will be apparent to theskilled worker from the previous discussion and need not be gone intofurther here.

It is presently impossible to assay a mixture the complexity of thosedescribed herein. However, by using the synthetic methods discussedbefore, a skilled worker can construct a mixed precursor oligopeptideset, which upon hydrolysis and amino acid analysis has molar ratios ofeach amino acid to each other in the range of about 0.5 to about 1.5;i.e., the molar ratio of one amino acid residue to any other residue is1:1±about 0.5, more preferably, this ratio is 1:1±about 0.25, whichratios carry through to the linear peralkylated oligopeptides.

Each chain of a set is also present in an equimolar amount and is of thesame length (contains the same number of peralkylated residues) comparedto the other chains present in the set. This equimolarity is alsoimpossible to measure directly. However, by carrying out each reactionto completion and maintaining the previously discussed equimolarity, onecan prepare chains that are of the same length and are present inequimolar amounts.

A precursor solid support-linked (-coupled) oligopeptide mixture set canalso be directly alkylated to form a solid support-linked peralkylatedoligopeptide set using a before-discussed reduction procedure.Alkylation while the peptide is linked to the support facilitatespurification of the resulting set from unwanted reaction products.

Such a support-linked set can then be used in an assay as discussedhereinafter for binding to a soluble reactor such as an antibody or anexternal cellular receptor such as ELAM-1, but is not as useful forgeneral assays for cellular receptors as is a free set.

It can also be useful for a peralkylated oligopeptide set to include alabel. A radioactive label such as ³ H can be used as part of anN-terminal acyl group of each member peralkylated oligopeptide.

Other contemplated labels include chromophores such as the2,4-dinitrophenyl or 4-nitrophenyl groups and fluorescent molecules suchas a dansyl group that can be coupled to an N-terminal amino group of aperalkylated oligopeptide using dansyl chloride(5-dimethylamino-l-naphthalenesulfonyl chloride).

A 2,4-dinitrophenyl or 4-nitrophenyl group can be coupled to anN-terminal amino group of a peralkylated oligopeptide set by means of anappropriate halogen derivative such as a chloro or fluoro group. Theresulting nitrophenyl aniline derivatives have a yellow to yellow/orangecolor that can be readily observed.

It is also contemplated that a photoreactive label be coupled to aperalkylated oligopeptide set, particularly at the N-terminus. Exemplaryphotoreactive labels include the 4-azidobenzoyl and 4-azidosalicylgroups that are present as N-terminal amides prepared by reaction of theN-hydroxysuccinimide ester of either group with the free, N-terminalamino group. Each of the esters is available from Sigma Chemical Co.,St. Louis, Mo.

Assay Processes and Peralkylated Oligopeptides

The present invention also contemplates a process for determining thesequence of a linear peralkylated oligopeptide ligand thatpreferentially (optimally) and specifically binds to an acceptor(receptor). Such a process can be carried out with the sets coupled tothe sold support used for synthesis or with those sets not coupled tothe solid support used for synthesis.

In accordance with one such process,

(a) a library of sets of linear peralkylated oligopeptides is providedin which each set comprises a mixture of equimolar amounts of linear C₁-C₇ -alkyl peralkylated oligopeptide member chains containing the samenumber of two to about ten peralkylated amino acid residues in eachperalkylated oligopeptide chain. Each peralkylated amino acid residueexcept proline has its peptidyl amino nitrogen atom alkylated with a C₁-C₇ -alkyl group. As discussed previously, the member chains of each sethave one or more (at least one) of at least six different predeterminedperalkylated amino acid residues at one or more (at least one)predetermined positions of the peralkylated oligopeptide chain, and eachset has equimolar amounts of at least six different peralkylated aminoacid residues at the same one or more (at least one) other positions ofthe peralkylated oligopeptide chain. Preferably, the same at least sixperalkylated residues are used at the mixture positions and thepredetermined position. However, in some instances, the one or morepredetermined positions of these sets are occupied by peralkylatedresidues not used in the mixture positions. The amino-terminus of eachof the peralkylated oligopeptides in the set is quaternaryalkylammonium, amino, N-alkylamino or a C₁ -C₁₈ hydrocarboylalkylamidogroup, and the carboxy-terminus is mono- or di-alkylamido,alkylcarboxylate or carboxyl group. A C₁ -C₇ -alkyl group at the N- orC-terminus is a C₁ -C₇ -alkyl group that can be the same or differentfrom the other C₁ -C₇ -alkyl groups present in each molecule. The setsof the library differ in that the one or more (at least one)predetermined peralkylated amino acid residues present at the one ormore (at least one) predetermined chain positions within each set isdifferent between the sets.

(b) Each set from the library of sets is separately admixed with theacceptor in an aqueous medium at a set concentration of about 0.1milligrams (mg) per liter to about 100 grams per liter, and preferablyabout 1 milligram per liter to about 100 grams per liter, and morepreferably about 100 milligrams per liter to about 20 grams per liter.The binding of each set to the acceptor is separately assayed, and theone or more sets of the plurality of sets that exhibits optimal orpreferential, specific binding compared to the other sets assayed isdetermined, thereby identifying the one or more peralkylated residuesthat provide optimal or preferential binding at that one or morepredetermined positions.

(c) A second library of sets of linear peralkylated oligopeptides isprovided in which each set comprises a mixture of equimolar amounts ofmember C₁ -C₇ -alkyl linear peralkylated oligopeptide chains containingthe same number of two to about ten peralkylated residues in eachperalkylated oligopeptide chain (having the same chain length) as thechains of first-named library of sets. Each peralkylated amino acidresidue except proline has its peptidyl amino nitrogen atom alkylatedwith a C₁ -C₇ -alkyl group. The members of each second library of setscontain the one or more peralkylated residues of the first library ofsets identified as exhibiting optimal or preferential, specific bindingin the one or more predetermined chain positions occupied in thefirst-named sets, and have one of at least six different predeterminedperalkylated amino acid residues at another preferably adjacentpredetermined position of the peralkylated oligopeptide chain differentfrom the position of the one or more predetermined positions of thefirst-named library of sets. Each of the second library of sets hasequimolar amounts of the same at least six different peralkylated aminoacid residues as the first-named sets at the same one or more otherpositions of the alkylated oligopeptide chain not occupied by the one ormore identified or predetermined peralkylated residues. The amino- andcarboxy-termini of the peralkylated oligopeptides of the second libraryof sets are the same as those of the first-named sets. The secondlibrary of sets thus differ from the first library of sets in that atleast two chain positions within the second set library are identifiedand predetermined (defined), and that second set library contains onefewer mixture positions than does the first set library.

(d) Each set from the second library of sets (of step c) is separatelyadmixed with the acceptor in an aqueous medium at a concentration ofabout 0.1 milligrams per liter to about 100 grams per liter andpreferably about 1 milligram per liter to about 100 grams per liter. Thebinding of each second library set to the acceptor is separately assayedand the one or more sets of the second library of sets that exhibitsoptimal or preferential, specific binding compared to the other setsassayed is determined, as discussed before, so that another peralkylatedresidue that provides optimal or preferential binding is determined.

(e) Steps (c) and (d) can be repeated with further, e.g., third, fourth,fifth, etc., libraries of sets until the desired number of setlibraries, e.g. two through seven, (typically at least three for a3-mer) have been assayed, each of those set libraries differing from theimmediately previous library by having one more defined (predetermined)position occupied by one of at least six predetermined peralkylatedresidues, and one fewer predetermined repeating unit position occupiedby equimolar amounts of at least six peralkylated residues. The repeatsof steps (c) and (d) can alternatively be repeated until the lastlibrary assayed did not exhibit an increase in preferential, specificbinding compared to the library assayed in the immediately precedingrepeated assay. If that is the case, the sequence can be determined.Typically, however, the process continues, and individual peralkylatedoligopeptides are prepared and assayed as discussed hereinafter. Forexample, the particular position assayed can be a position of redundancywithin a longer sequence whose other as yet undefined positions, oncedefined, are needed for binding. A better binding or otherwisesynthetically convenient residue is then used at the position wherepreferential binding was not exhibited.

Each of those further libraries of sets of linear peralkylatedoligopeptides of (e) comprises a mixture of equimolar amounts of memberlinear C₁ -C₇ -alkyl peralkylated oligopeptide chains containing thesame number of two to about ten peralkylated amino acid residues in eachperalkylated oligopeptide chain as the chains of the first-named libraryof sets. Each peralkylated amino acid residue except proline has itspeptidyl amino nitrogen atom alkylated with a C₁ -C₇ -alkyl group. Themember chains of each further library of sets contain the peralkylatedamino acid residues in the peralkylated oligopeptide chain positionsthat exhibited preferential, specific binding in a library of sets usedimmediately before and also one of at least six different predeterminedperalkylated amino acid residues at another preferably adjacentpredetermined position of the peralkylated oligopeptide chain that isdifferent from the positions of the identified peralkylated amino acidresidues of the library of sets used immediately before. Thus, eachsubsequent library of sets contains each of the previously identifiedperalkylated residues in the peralkylated oligopeptide chain positionthat exhibited preferential, specific binding, as well as a preferablyadjacent predetermined peralkylated residue at a position in theperalkylated oligopeptide chain previously occupied by an equimolarmixture position. Each of those further library member sets also has thesame termini as the first-named sets and has equimolar amounts of the atleast six different peralkylated amino acid residues of said first-namedsets at the same one or more positions of the peralkylated oligopeptidechain not occupied by the identified peralkylated amino acid residues orthe predetermined peralkylated amino acid residues.

As noted previously, it is preferred that the one or more predeterminedpositions of the libraries of (a) are at one or the other terminus ofthe peralkylated oligopeptide chain, more preferably the N-terminus. Itis also preferred that each new predetermined position in subsequentlyused sets be in a position adjacent to the position whose peralkylatedamino acid residue was identified in the immediately previous assay.Thus, as each of steps (c) and (d) is repeated with new libraries ofsets, one more position in the sequence becomes identified, and the setscontain one fewer mixture position.

In usual practice, once the preferential or optimal, specific bindingperalkylated residues for all but the last position have beendetermined, at least six individual linear peralkylated oligopeptidechains are provided. These molecules contain the same number of C₁ -C₇-alkyl peralkylated residues and same termini as did the chains of thefirst-named library of sets, and contain the peralkylated amino acidresidues in the sequence determined by the above assays; i.e., themolecules contain each of the identified peralkylated residues at itsposition that exhibited preferential binding in the previous assays, andone each of the at least six peralkylated amino acid residues used atthe final position. These at least six peralkylated oligopeptides areseparately admixed with the acceptor and assayed for preferential oroptimal, specific binding as discussed before. Determination of theperalkylated residue that exhibits preferential, specific binding ascompared to the other peralkylated residues assayed from the results ofthis group of assays provides the last peralkylated residue of thesequence and thereby a preferential binding sequence for the linearperalkylated oligopeptide.

In usual practice, a peralkylated oligopeptide length; i.e., number ofperalkylated residues in the chain, is selected and a complete sequenceof a preferential, specific binding peralkylated oligopeptide isdetermined. In some instances, as noted before, preferential, specificbinding does not increase when a further library is assayed; i.e., asadditional known peralkylated residues are used in place of mixtures ofperalkylated residues. In the latter case, the preferential, specificbinding sequence is thus determined and it is unnecessary to utilize theat least six individual peralkylated oligopeptides as discussed above.If preferential, specific binding increases when each further library isused and assayed, the individual peralkylated oligopeptides discussedabove are prepared and used.

The above assay process is particularly useful with sets prepared fromthe before-discussed 400 corresponding 6-mer oligopeptide sets in whichtwo positions are of known sequence. Thus, after the first assay, thetwo N-terminal preferential, specific binding peralkylated residues aredetermined. In step (d), the third position is scanned for preferential,specific binding. This process is continued until the sequence of theN-terminal five peralkylated residues is known. Individual peralkylatedoligopeptides are then usually used to complete the determination of theoverall preferential, specific binding sequence by determiningpreferential, specific binding for the last position of this exemplary6-mer.

Also preferred are sets and set libraries that are 5-10 peralkylatedresidues in length whose corresponding C-terminal four positions areoccupied by peralkylated amino acid residue side chain mixtures, andwhose corresponding amino-terminal positions are occupied bypredetermined peralkylated residues. Each above set can be prepared froma single preparation of solid support-coupled 4-mer oligopeptidemixtures to which one or more predetermined acid residues is coupled,then peralkylated and cleaved following each acceptor binding assay.

For example, starting with a batch of support-coupled 4-mer oligopeptidemixtures whose positions are all equimolar mixtures, twenty mixtures canbe prepared by separately coupling each of the twenty natural aminoacids to a separate portion of the batch. After peralkylation of theprecursor solid support-coupled mixture pool so prepared and cleavage, abinding assay is run as with a monoclonal antibody to determinepreferential binding. Another library of twenty sets is then preparedusing the same 4-mer batch with an optimal binding residue (afterperalkylation) at position 2 in the sequence from the correspondingN-terminus and each of the twenty residues at position 1. The bindingassay is run again after peralkylation and cleavage, and optimal bindingis determined. This process is continued until a predeterminedperalkylated oligopeptide sequence of desired length is completed. Theuse of C-terminal mixture positions that remain mixtures throughouthelps improve the solubility of the set.

Another particularly preferred assay process utilizes library setsprepared from precursor positional oligopeptide sets, such as thelibrary of 120 precursor 6-mer sets discussed before. Here,

(a) a library of sets of linear peralkylated amino acidresidue-containing oligopeptides in which each library comprises amixture of equimolar amounts of C₁ -C₇ -alkyl linear peralkylatedoligopeptide chains containing the same number of two to tenperalkylated residues in each peralkylated oligopeptide chain isprovided. As discussed previously, the members of each set of thelibrary have one of at least six different predetermined peralkylatedamino acid residues at a single predetermined repeating unit position ofthe peralkylated oligopeptide chain, and N- and C-termini as discussedbefore. Each peralkylated amino acid residue except proline has itspeptidyl amido nitrogen atom alkylated with a C₁ -C₇ -alkyl group. Eachset has equimolar amounts of those same at least six differentperalkylated amino acid residues at the same other positions of theperalkylated oligopeptide chain. As is the case with all of the setsdiscussed herein, it is preferred to use about 10 or more and morepreferable to use about 15 to about 20 different peralkylated amino acidresidues. A C₁ -C₇ -alkyl group at the N- or C-terminus is a C₁ -C₇-alkyl group that can be the same or different from the other C₁ -C₇-alkyl groups present in each molecule. The sets of the library differin that the single predetermined peralkylated amino acid residue presentat a single predetermined chain position within each set is differentbetween the sets.

(b) Each set from the library of sets is separately admixed with theacceptor in an aqueous medium at a set concentration of about 0.1milligrams per liter to about 100 grams per liter, and preferably about1 milligram per liter to about 100 grams per liter. The binding of eachset to the acceptor is separately assayed, and the one or more sets ofthe library of sets that exhibits preferential, specific bindingcompared to the other sets assayed is determined, thereby identifyingthe one or more peralkylated residues that provide preferential bindingat that single, predetermined position.

(c) A second library of sets of linear peralkylated oligopeptides isprovided in which each set comprises a mixture of equimolar amounts oflinear C₁ -C₇ -alkyl peralkylated oligopeptide chains containing thesame number of two to ten peralkylated residues in each peralkylatedoligopeptide chain (having the same chain length) as the first-namedlibrary of sets. Each peralkylated amino acid residue except proline hasits peptidyl amido nitrogen atom alkylated with a C₁ -C₇ -alkyl group.The members of each second library set have the same N- and C-termini asthe first library set member chains, and have one of the same at leastsix different predetermined peralkylated amino acid residues of thefirst-named sets at another single predetermined position of theperalkylated oligopeptide chain different from the position of thefirst-named library of sets, and each of these sets has equimolaramounts of the same at least six different peralkylated amino acidresidues at the same other positions of the peralkylated oligopeptidechain. The second library of sets differs from the first library of setsin that the single predetermined chain position within each set thatcontains the one of at least six different peralkylated residues isdifferent between the libraries.

Put differently, the second library of sets has the same length andtermini as the first-named library, and has equimolar amounts of the atleast six peralkylated residues at the peralkylated oligopeptide chainposition occupied by the single peralkylated residue in the first-namedset library, and a single peralkylated residue in a position occupied byequimolar amounts of peralkylated residues in the first-named librarysets. For example, the first named library of sets can have its singleone of at least six different predetermined peralkylated amino acidresidues at position 1 with the other positions occupied by mixtures,whereas this second library of sets has its single predeterminedperalkylated amino acid residues at any of positions 2-10, and equimolaramounts of at least six different peralkylated residues at position 1,and the remaining chain positions other than that occupied by the singleperalkylated residue.

(d) Each set from the second library of sets (of step c) is separatelyadmixed with the acceptor in an aqueous medium at a concentration ofabout 0.1 milligrams per liter to about 100 grams per liter andpreferably about 1 milligram per liter to about 100 grams per liter. Thebinding of each second set to the acceptor is separately assayed and theone or more sets of this second library of sets that exhibitspreferential, specific binding compared to the other sets assayed isdetermined, thereby identifying a peralkylated amino acid residue thatprovides preferential binding at that predetermined position in theperalkylated oligopeptide chain.

(e) Steps (c) and (d) are repeated with third, fourth, fifth, etc. up tothe number of residues in the chain length, libraries of sets until thedesired number of set libraries have been assayed, each of those setlibraries differing from the other libraries by the position thatcontains the one of at least six different peralkylated amino acidresidues. It should also be apparent that where peralkylated dipeptidesare used, the process is stopped so that steps (c) and (d) can berepeated zero times.

The identity and position of the C₁ -C₇ -alkyl peralkylated amino acidresidue of each one or more sets that provided preferential or optimal,specific binding so determined for each library of sets provides theidentity of a peralkylated residue sequence for the ligand thatpreferentially, specifically binds to the acceptor. Thus, because eachof the libraries of positional sets assayed provides the identity of aperalkylated residue(s) that provide(s) enhanced binding for thatposition, and because there is equimolar representation of all the otherperalkylated residues at the mixture positions, knowledge of theidentity and position of peralkylated residues that provide enhancedbinding for the utilized positions provides a sequence for a ligand ordonor-peralkylated oligopeptide that provides enhanced binding.

It should be understood that determining the identity and position oftwo peralkylated residues that each provide greatly enhanced binding canbe extremely useful when preparing completed peralkylated peptidesbecause several fewer such peralkylated peptides need be prepared. Ofcourse, knowledge of three identities and positions is more preferred,and knowledge of four is more preferred still, etc.

The above process is referred to as a scanning process in thatperalkylated residues at each position of a sequence are individuallyscanned.

It is preferred that, as a group, the single, predetermined repeatingunit positions be adjacent to each other. Thus, exemplary sets forpositions 1-3 of a trimer, or positions 2-6 of a hexamer are used or 1-6of a decamer peralkylated oligopeptide are used, as compared topositions 1, 2 and 4-8 of an octamer.

It should be understood that although it is preferred to scan adjacentrepeating unit positions, one need not utilize the pluralities of setsin any order by position. Thus, although convenient, one need not usethe libraries of sets that contain the one of at least six differentpredetermined peralkylated residues at position 1 followed by thelibraries having the one of at least six different predetermined sidechains at position 2, and so on.

In addition to there being no need to utilize the libraries ofpositional sets in any order, it is also not necessary to utilize asingle library of positional sets followed by another and another, etc.Rather, one can utilize the individual sets in any order because theposition and identity of the single one of at least six differentpredetermined peralkylated amino acid residues of each library is known.This is in contrast to the previously discussed process where it ispreferred to use a predetermined peralkylated residue adjacent to anidentified peralkylated residue.

Thus, a more general scanning process is also contemplated. Here, (a) alibrary or plurality of sets of linear peralkylated oligopeptides isprovided. Each set of that library or plurality of sets comprises amixture of equimolar amounts of linear C₁ -C₇ -alkyl peralkylatedoligopeptide chains containing the same number of two to about tenperalkylated residues in each chain, and having N- and C-termini asdiscussed before. Each peralkylated amino acid residue except prolinehas its peptidyl amido nitrogen atom alkylated with a C₁ -C₇ -alkylgroup. Each set has a single one of at least six different predeterminedperalkylated amino acid residues at a single, predetermined position ofthe peralkylated oligopeptide chain, and has equimolar amounts of eachof the same at least six different peralkylated amino acid residues atthe same other positions of the peralkylated oligopeptide chain. A C₁-C₇ -alkyl group at the N- or C-terminus is a C₁ -C₇ -alkyl group thatcan be the same or different from the other C₁ -C₇ -alkyl groups presentin each molecule. Each set differs from the other sets in that theidentity and chain position of the one of at least six differentpredetermined peralkylated amino acid residues present at the singlepredetermined chain position within each set is different between thesets. The maximum number of sets provided is equal to the product of thenumber of different amino acid residues present at the predeterminedchain positions containing the one of at least six different residuestimes the number of different chain positions containing the one of atleast six different predetermined peralkylated amino acid residues.

(b) Each set is separately admixed with the acceptor in an aqueousmedium at a set concentration of about 0.1 milligrams per liter to about100 grams per liter and preferably about 1 milligram per liter to about100 grams per liter. The binding of each set to the acceptor isseparately assayed for each set. The one or more sets that providepreferential, specific binding for each different chain position isdetermined.

The identity and position of the C₁ -C₇ -alkyl peralkylated residue ofeach one or more sets that exhibited preferential, specific bindingprovides the peralkylated residue sequence for the ligand thatpreferentially binds to the acceptor.

Although an above process can be carried out with peralkylated dipeptidesets, it is preferred to use sets of at least pentamers. Thus, at leastfive libraries of positional sets are typically utilized (scanned). Itis preferred, but not necessary, that those five libraries of sets, as agroup, contain single, predetermined peralkylated residues at adjacentpositions in the sequence. For example, in a 5-mer, those positionswould be 1-5 of the sequence. However, in a 10-mer, those positionscould be positions 6-10, 5-9, 3-7 or the like. Of course, one obtainsmore precise sequence identification information if adjacent positionsof the peralkylated oligopeptide chain are determined, and if theidentity of the peralkylated residue exhibiting enhanced binding foreach chain position is determined.

Those identified C₁ -C₇ -alkyl peralkylated residues that exhibitpreferential binding within about a factor of two of a best binding sidechain at that position are typically considered to exhibit preferentialor optimal, specific binding and are used to prepare one or moreperalkylated oligopeptides using the other identified peralkylatedresidues at the other positions to determine which combination providesoptimal or preferential overall properties. Thus, using a 6-mer asexemplary, although one may not be able to determine a single optimalsequence out of the 64,000,000, the field is typically cut down to about5-50 or sometimes thousands of sequences, which because of theirsequential similarity, can be readily prepared by the SMPS methoddiscussed in U.S. Pat. No. 4,631,211, followed by reduction. Even wherethe scanning process narrows the possible optimal binding linearperalkylated oligopeptide sequences to several thousand, the worker'sknowledge has been advanced, and he or she can use a peptide synthesismethod described in WO 92/09300, or Houghten et al., Nature, 354:84(1991), in U.S. Pat. Nos. 5,010,175, 5,182,366 or 5,144,392 followed byperalkylation to complete the sequence or obtain new optimal bindingsequences.

In any assay discussed herein, all of the at least six differentpredetermined peralkylated residues at a predetermined position canprovide similar specific binding. That phenomenon is referred to aspositional redundancy or redundancy, and any convenient peralkylatedresidue is utilized at that position when a peralkylated oligopeptideligand is synthesized.

The aqueous medium used in an assay can be extremely varied and includestap water, distilled or deionized water, as well as a buffer solution asis used for antibody binding studies or a cell growth medium as isuseful for culturing bacteria, yeast, fungi, plant or animal cells, allof which are well known to skilled workers.

The concentration of a linear peralkylated oligopeptide set in theaqueous medium is selected so that the peralkylated oligopeptide set ispresent at concentrations of about 0.1 milligrams per liter to about 100grams per liter, preferably about 1.0 μg/ml to about 100 mg/ml, and morepreferably about 0.1 mg/ml to about 20 mg/ml. Thus, when eachperalkylated oligopeptide mixture is made up of 3.2 million individualperalkylated oligopeptides; e.g. a permethylated C--N-methylamide 6-meroligopeptide using the 20 natural amino acid residues, then each 6-merperalkylated oligopeptide within each mixture is present in a preferredconcentration of about 1.0 μg/ml/3,200,000=0.31 pg/ml, to about 100mg/ml/3,200,000=31.25 ng/ml. Presuming an average molecular weight of apermethylated C-terminal N-methylamide 6-mer peralkylated peptide to beabout 800 g/mole, then at 1.0 μg/ml, the individual hexamers are presentat a concentration of about 0.4 pmolar and at 100 mg/ml the individualhexamers are present at about 40 nmolar. Most preferably, concentrationsof about 0.5 mg/ml to about 10 mg/ml are used.

It is preferred, although not required, that a peralkylated oligopeptidemixture set be soluble in the aqueous medium utilized. Thus, thepermethylated N-terminal quaternized oligopeptide sets are typicallysoluble in most aqueous media, whereas sets alkylated with morehydrophobic alkyl groups form milky dispersions. Such dispersions arenonetheless useful. For example, the free N-terminal amino perbenzylatedsingle peptide GlyGlyPheLeu SEQ ID NO:36 formed an opaque dispersion inwater containing 10 volume percent DMSO at 1 mg/ml. Similar dispersionswere prepared from peralkylations with ethyl and allyl groups.

As is well known, complete water-solubility is not required for a drug,as many commonly used drugs such as steroids are substantially insolublein water, aqueous assay media or body fluids, but are nonethelessextremely valued medicaments. For example, the Merck Index, 10th ed.,lists both progesterone and testosterone as being insoluble in water andestradiol as being almost insoluble in water.

It is to be understood that the wide breadth of concentrations specifiedabove is intended to take into account the contemplated range ofperalkylated oligopeptide sets that can have up to nine positions asmixtures, one to about four alkyl groups per residue, alkyl groups ofvarying molecular weight and the fact that wide ranges of concentrationsare often used for determining IC₅₀ and K_(i) values.

A peralkylated oligopeptide set and its individual members can be lookedat as donor (ligand) in donor-acceptor (receptor) binding complexformation. Exemplary acceptor molecules are antibody combiningsite-containing molecules such as whole antibodies, F(ab), F(ab')₂ andFv antibody portions, solubilized or non-solubilized cell surfacereceptor molecules, such as the solubilized CD4 receptor, internalcellular receptors and viral protein receptors, all but the antibodycombining site-containing molecules being collectively referred to as"cellular receptors". "Cellular receptors" also include living cellsthat contain receptors that interact with a peralkylated oligopeptidelibrary as ligand (donor).

The binding interactions assayed for here are specific bindinginteractions such as those between antibody and antigen. Such specificbinding interactions are to be compared to the non-specific binding thatis observed between most proteins and the plastics used in microtiterplates.

Any well known binding or binding inhibition assay format can be used.For example, a solid phase assay using a solid phase-bound antibodybinding site and a radiolabeled peralkylated oligopeptide set iscontemplated. Also contemplated is a competitive binding assay in whicha protein or polypeptide is bound to a solid phase as an antigen and amonoclonal antibody binding to that antigen is admixed with aperalkylated oligopeptide set. Inhibition of binding of the monoclonalantibody by the peralkylated oligopeptide set provides a measure of thebinding between the peralkylated oligopeptides and monoclonal antibody.Monoclonal antibody binding inhibition and the inhibition of otheracceptors' binding can be assayed using enzyme or radiolabels as is wellknown.

It is often the case that one has receptors (acceptors) such asantibodies to a particular ligand such as an antigen, but the specificligand (antigen) that binds those antibodies is unknown. Under thesecircumstances, usual solid phase assays in which the ligand is affixedto a plate or other solid phase matrix cannot be carried out because therelatively short peralkylated oligopeptide sets contemplated herein donot bind well to microtiter plate walls and similar solid phasematrices.

Avidin binds well to microtiter plate walls and similar matrices. Use ofthat fact and its well known binding partner, biotin, can be made forthose assays in which the ligand bond by the receptor is unknown or isotherwise unavailable.

Thus, avidin is coated on a solid phase matrix such as microtiter platewalls using standard, well known techniques such as adsorption. Biotin,which contains a free carboxyl group, is coupled to the N-terminal amineof a before-described peralkylated oligopeptide set via the biotincarboxyl group, using usual coupling chemistry as described herein forcoupling amino acids. The biotinylated set is dissolved in an aqueousmedium and admixed with the avidin-coated solid phase matrix to form asolid/liquid phase admixture. That admixture is maintained for a timeperiod sufficient for the avidin and biotinylated peralkylatedoligopeptide set complex, typically five minutes to about five hours,and form a biotinylated peralkylated oligopeptide set-containing solidsupport and a liquid phase depleted of biotinylated peralkylatedoligopeptide. The solid and liquid phases are then separated, and thesolid support is typically washed.

The thus prepared solid support that contains an affixed peralkylatedoligopeptide set, is then utilized with the receptor (acceptor) instandard solid phase assays. Where the receptor is an antibody, usualdetecting systems such as the use of radiolabeled or enzyme-linkedanti-antibodies such as goat anti-mouse antibodies where the receptorsare mouse antibodies are utilized to detect binding. Where the receptoris a cellular receptor, radiolabels incorporated into the receptor byculture of the cells in a medium containing radioactive amino acids aretypical detecting means of choice.

It is frequently convenient to provide a spacer group between theperalkylated oligopeptides of a set and the biotin. Exemplary spacersinclude one to about five glycine, C₂ -C₆ straight chain ω-amino acidssuch as glycine, α-alanine, 4-aminobutyric acid (GABA) or 4-aminocaproicacid.

Thus, a N-terminal biotinylated peralkylated oligopeptide set asotherwise described before is also contemplated. That biotinylatedperalkylated oligopeptide set can further include one to about five C₂-C₆ straight chain ω-amino acid residues between the N-terminal amine ofthe peralkylated oligopeptides and the biotin group.

For a before-discussed chromophore- or fluorescent-labeled peralkylatedoligopeptide set, contact between the acceptor and peralkylatedoligopeptide set can be carried out with the acceptor linked to a solidsupport such as sepharose or agarose.

The non-binding and poorer binding sets can be separated from the solidsupport-bound acceptor molecules by washing at increasingly higher saltconcentrations until a predetermined concentration is reached that isused to define a better or preferential binding peralkylatedoligopeptide. The choromophoric or fluorescent label can be used tofollow the elution. Using the 2,4-dinitrophenyl chromophore asexemplary, the presence of a yellow to yellow/orange color on the solidsupport for a given set after washing indicates an optimal binding set.

An exemplary assay using a photoreactive label can be carried out withan enzyme having a known substrate. Here, the enzyme as acceptor andphotoreactive labeled, peralkylated oligopeptide set are admixed and theadmixture maintained so that binding can occur. The admixture is thenirradiated using sufficient quanta of light at an appropriatewavelength, as are well known, to cause the decomposition of thephotoreactive group such as an azide group and the insertion of theresulting peralkylated oligopeptide containing radical into the enzymepolypeptide backbone. That insertion links the peralkylated oligopeptideto the enzyme and blocks reaction with the enzyme's substrate. Thus, anassay of enzymic activity after irradiation provides a determination ofwhich peralkylated oligopeptide set bound optimally, with a diminishedactivity indicating enhanced binding.

Cellular receptor molecules are also particularly contemplated as usefulin this assay system. The cellular receptor whose binding iscontemplated for assay need not be isolated, but can be part of anintact, living cell such as bacterial, yeast, fungal, mammalian or plantcells, or viruses. When such intact, living cells are utilized, relativebinding amounts can be determined by the growth or inhibition of growthof the admixed, assayed cells. The aqueous medium here is a growth orculture medium, known to promote growth of the assayed cells.

The concentration of free acceptor molecules, including those obtainedfrom cell preparations or those present in intact, living cells used forsuch binding assays is an assay-effective amount such as is normallyused for such assays, and is well known in the art. It is to beunderstood that different concentrations of free acceptor molecules orthose present in intact, living cells can vary with each acceptorstudied.

A before-described assay can be carried out in vitro as well as beingcarried out in vivo. For in vivo assays, living plants such as tobacco,alfalfa, corn (maize), zinnias and the like are contemplated hosts,whereas small laboratory mammals such as rats, mice, guinea pigs,rabbits and dogs are contemplated hosts for animal assays.

A C₁ -C₇ -alkyl peralkylated oligopeptide set-containing composition canbe administered and a C₁ -C₇ -alkyl peralkylated oligopeptide contactedwith the acceptors internally or externally in plants through watering,misting of foliage, or injection. For the animals, a composition can beadministered internally, orally or by injection such asintraperitoneally, subcutaneously or intramuscularly or topically as byapplication to skin for the contact between donor and acceptor to takeplace.

Binding here can be assessed by relative growth rate (positive ornegative) or by the affect of the composition on one or more tissues, asthrough microscopic examination, by body temperature wherepathogen-infected animals are used, and the like as are well known.

Peralkylated Oligopeptides

Several peralkylated individual oligopeptides were prepared based on thestudies discussed hereinafter. Ten permethylated hexamers, a pentamer,heptamer and octamer were found to be particularly and selectivelyactive against Gram-positive bacteria Staphylococcus (S.) aureus, amethicillin-resistant strain of S. Aureus, and Streptoccus (S.)sanquis!, while having substantially no activity against E. coli,Gram-negative bacteria, or Candida albicans, a yeast; IC_(50's) >600μg/ml. These permethylated oligopeptides also exhibited substantially nolysis of human red blood cells at concentrations equal to their IC₅₀values, and no to minor amounts of lysis at a concentration about 10- toabout 100-fold greater than their IC₅₀ values; i.e., at 350 μg/ml. Anabsence of in vitro toxicity toward McCoy cells (ATCC 1696-CRL) using anMTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;Mosmann, J. Immunol. Methods, 65:55-63 (1983)! assay was also noted forthese materials. These facts, coupled with the observed stability ofpermethylated peptides to proteolytic enzymes, the excellentwater-solubility exhibited by the peptides and the favorableaqueous/organic solvent partitioning enhance the value of thesepermethylated oligopeptides as Gram-positive antibacterial agents.

These particularly preferred permethylated oligopeptides are listedbelow using the prefix "PerM-" to indicate permethylation. Eachpermethylated oligopeptide contains a C-terminal N-methylamide, that isnot shown and an N-terminal trimethylammonium group that is also notshown.

    PerM-LeuPheIlePhePhePhe                                    (SEQ ID NO:26);

    PerM-PhePheIlePhePhePhe                                    (SEQ ID NO:27);

    PerM-PhePhePhePhePhePhe                                    (SEQ ID NO:23);

    PerM-LeuPhePhePhePhePhe                                    (SEQ ID NO:28);

    PerM-PhePhePhePheHisPhe                                    (SEQ ID NO:29);

    PerM-LeuPheIlePhePheHis                                    (SEQ ID NO:30);

    PerM-LeuPhePhePheHisPhe                                    (SEQ ID NO:31);

    PerM-LeuPheIlePheHisPhe                                    (SEQ ID NO:32);

    PerM-LeuPhePhePhePheHis                                    (SEQ ID NO:33);

    PerM-PhePheIlePhePheHis                                    (SEQ ID NO:34);

    PerM-PhePhePhePhePhe                                       (SEQ ID NO:22)

    PerM-PhePhePhePhePhePhePhe                                 (SEQ ID NO:24);

and

    PerM-PhePhePhePhePhePhePhePhe                              (SEQ ID NO:25).

The first ten permethylated oligopeptides correspond to the formula

    Xaa.sub.1 PheXaa.sub.3 PheXaa.sub.5 Xaa.sub.6,             (SEQ ID NO:35)

wherein Xaa₁ at the first position is a N-trimethylammonium Leu or Pheresidue;

Xaa₃ at the third position is Ile or Phe;

Xaa₅ at the fifth position is His or Phe; and

Xaa₆ at the sixth position is a C-terminal

N-methylcarboxamido His or Phe residue, with the proviso that at leastone of Xaa₅ and Xaa₆ is Phe. When each of Xaa₁, Xaa₃ Xaa₅ and Xaa₆ isPhe, a permethylated peptide of SEQ ID NO:23 results.

The above list also illustrates that permethylated C-terminalN-methylamido oligophenylalanine (oligophe) having 5 through 8 Pheresidues are contemplated. These permethylated oligopeptides also havean α-N-terminal trimethylammonium group. Of these permethylated oligopheC-terminal N-methylamido peptides, compounds having 6, 7 or 8 Pheresidues are most preferred; i.e., SEQ ID NO's: 23, 24 and 25.

The following examples are intended to illustrate, but not limit, theinvention.

EXAMPLE 1 Exemplary Synthesis of a Set of Mixed Oligopeptides havingEquimolar Amounts of the Twenty Natural Amino Acid Residues

Aliquots of five grams (4.65 mmols) of p-methylbenzhydrylaminehydrochloride resin (MBHA) are placed into twenty porous polypropylenebags. These bags are placed into a common container and washed with 1.0liter of CH₂ Cl₂ three times (three minutes each time), then againwashed three times (three minutes each time) with 1.0 liter of 5 percentDIEA/CH₂ Cl₂ (DIEA=di-isopropylethylamine; CH₂ Cl₂ =DCM) . The bags arethen rinsed with DCM and placed into separate reaction vessels eachcontaining 50 ml (0.56M) of the respective t-BOC-amino acid/DCM.N,N-Diisopropylcarbodiimide (DIPCDI; 25 ml; 1.12M) is added to eachcontainer, as a coupling agent.

Twenty amino acid derivatives are separately coupled to the resin in50/50 (v/v) DMF/DCM. After one hour of vigorous shaking, Gisen's picricacid test Gisen, Anal. Chem. Acta, 58:248-249 (1972)! is performed todetermine the completeness of the coupling reaction. On confirmingcompleteness of reaction, all of the resin packets are then washed with1.5 liters of DMF and washed two more times with 1.5 liters of CH₂ Cl₂.

After rinsing, the resins are removed from their separate packets andadmixed together to form a pool in a common bag. The resulting resinmixture is then dried and weighed, divided again into 20 equal portions(aliquots), and placed into 20 further polypropylene bags (enclosed). Ina common reaction vessel the following steps are carried out: (1)deprotection is carried out on the enclosed aliquots for thirty minuteswith 1.5 liters of 55 percent TFA/DCM; and 2) neutralization is carriedout with three washes of 1.5 liters each of 5 percent DIEA/DCM.

Each bag is placed in a separate solution of activated t-BOC-amino acidderivative and the coupling reaction carried out to completion asbefore. All coupling reactions are monitored using the abovequantitative picric acid assay. Next, the bags are opened and theresulting t-BOC-protected dipeptide resins are mixed together to form apool, aliquots are made from the pool, the aliquots are enclosed,deprotected and further reactions are carried out.

This process can be repeated any number of times yielding at each stepan equimolar representation of the desired number of amino acid residuesin the peptide chain. The principal process steps are convenientlyreferred to as a divide-couple-recombine (DCR) synthesis.

After a desired number of such couplings and mixtures are carried out,the polypropylene bags are kept separated to here provide the twentysets having the amino-terminal residue as the single, predeterminedresidue, with, for example, positions 2-4 being occupied by equimolaramounts of the twenty residues. To prepare sets having the single,predetermined amino acid residue at other than the amino-terminus, thecontents of the bags are not mixed after adding a residue at thedesired, predetermined position. Rather, the contents of each of thetwenty bags are separated into 20 aliquots, deprotected and thenseparately reacted with the twenty amino acid derivatives. The contentsof each set of twenty bags thus produced are thereafter mixed andtreated as before-described until the desired oligopeptide length isachieved.

The side chain protecting groups used with a-amino-terminal t-BOC andFmoc protecting groups are usually different. The side chain protectinggroups utilized for one type of synthesis or the other are as shown inthe table below. Other usually used side chain protecting groups arealso utilized for both types of syntheses.

    ______________________________________                                        Side Chain Protecting Group                                                   Amino Acid    N-t-BOC        N-Fmoc                                           Derivative    Protected      Protected                                        ______________________________________                                        Arginine      Toluenesulfonyl*                                                                             Mtr**                                            Cysteine      p-Methoxybenzyl                                                                              t-Butyl ether                                    Glutamic acid O-Benzyl       t-Butyl ester                                    Histidine     N-im-dinitrophenyl*                                                                          Trityl                                           Lysine        N-(o-chlorobenzyl-                                                                           t-BOC                                                          oxycarbonyl)                                                    Serine        O-Benzyl       t-Butyl ether                                    Threonine     O-Benzyl       t-Butyl ether                                    Tyrosine      O-(m-bromobenzenyl-                                                                          t-Butyl ether                                                  oxycarbonyl)                                                    Aspartic acid O-Benzyl       t-Butyl ester                                    ______________________________________                                         *Arginine and histidine are coupled in the presence of                        Nhydroxylbenztriazole  Hruby et al., Angew. Chem. Int. Ed. Engl.,             10:336-339 (1971)!.                                                           **Mtr = 4Methoxy-2,3,6-trimethylbenzenesulfonyl.                         

For precursor oligopeptide mixture sets not having an N-terminal C₁ -C₁₈acyl (e.g. acetyl) group, the following procedure is used for side chaindeprotection of N-t-BOC-protected oligopeptide chains. The fullyprotected solid support-coupled oligopeptide mixtures are treated with55 percent trifluoroacetic acid in methylene chloride prior to the HFtreatment to remove the final t-BOC-protecting group. Then the protectedsolid support-coupled oligopeptide mixtures, in polypropylene meshpackets Houghten, Proc. Natl. Acad. Sci., USA, 82:5131-5135 (1985)! arerinsed with alternating washes of DCM and isopropanol, and dried underreduced pressure for twenty-four hours.

Where cleavage from the solid support is carried out prior toalkylation, the low HF step Tam et al., J. Am. Chem. Soc., 195:6442-6455(1983)! is carried out in a two liter polypropylene reaction vessel,using a solution of 60 percent dimethylsulfide, 25 percent HF, 10percent p-cresol and 5 percent ethylenedithiol. The ethanedithiol isused to cleave the N-formyl groups from tryptophan residues. Where it isdesired not to cleave the N-formyl groups, ethanedithiol is omitted fromthe mixture and its amount is replaced by HF. N.sub.α -t-BOC-N-formyltryptophan is available from Bachem, Inc., Torrence, Calif.

HF is condensed at -78° C. After condensation, the HF-scavenger solutionis carefully transferred to the reaction vessel that contained theresin-containing packets. The low HF solution is made to give 5 ml per0.1 mmol of oligopeptide. After the reagents are added, the reactionvessel is placed in an ice water bath and shaken for two hours. The lowHF solution is removed and the packets containing the deprotectedpeptide resins are quickly washed with chilled DCM. The DCM wash isrepeated nine times (one minute each) followed by ten alternating washesof isopropanol and DCM. Finally, the resin is washed five times withDMF, then twice more with DCM. Deprotected peptide resin packets aredried under reduced pressure. After this process is completed, theunprotected peptides are ready to be cleaved by anhydrous HF.

The N-terminal Fmoc protecting groups of enclosed, protected solidsupport-coupled oligopeptide mixtures are removed by treatment withtwenty percent piperidine in DMF for ten minutes. Then the resultingN-deprotected, side chain-protected peptide resins in polypropylenepackets are washed with DMF twice (five minutes each) followed by tworinses with DCM (one minute each) and dried in a vacuum for twenty-fourhours.

The side chain deprotection is carried out in a two liter polypropylenereaction vessel, using a solution of 85 percent TFA, 5 percent phenol, 4percent thioanisole, 4 percent deionized H₂ 0 and 2 percentethanedithiol. The resins are shaken for 3.5 hours at room temperature.The reaction solution is removed, and the packets containing thecompletely deprotected solid support-coupled oligopeptide mixtures arequickly washed with chilled ether. The ether wash is repeated nine times(one minute each) followed by ten alternating washes of isopropanol andDCM. Finally, the solid support-coupled oligopeptide mixtures are washedfive times with DMF, then twice more with DCM. Deprotected solidsupport-coupled oligopeptide mixtures and their enclosing packets aredried under reduced pressure. After this process is completed, theunprotected peptides are ready to be cleaved by anhydrous HF.

Where an N-acyl group such as an acetyl group is to be present on anoligopeptide mixture set, the final t-BOC or Fmoc protecting group isremoved as above, an excess of acetic anhydride is added and thereaction is maintained until there are no more free amino groups presentas discussed elsewhere herein. The above rinsing and drying steps arethen carried out, followed by deprotection and cleavage of theoligopeptide mixture set from the solid support.

As noted earlier for post cleavage peralkylations, use of abenzhydrylamine resin as a solid support and anhydrous HF/anisole forcleavage of the oligopeptide mixture set provides a C-terminal amidogroup for the oligopeptide mixture set produced. Use of an ester-linkedresin solid support and that cleavage procedure provides a C-terminalcarboxylic acid. Use of a disulfide-containing linking group between thesolid support and oligopeptide chains as discussed in U.S. Pat. No.4,031,211 and cleavage with a disulfide bond breaking agent as discussedprovides a C-terminal mercaptan linking group amide-bonded to theoligopeptide chains. Subsequent peralkylation provides dialkylamido,alkyl ester and alkyl thioether C-terminal groups.

In an exemplary preferred peralkylation, more than one peptide-coupledsolid support (resin) is permethylated at one time by enclosing eachpeptide-coupled resin in separate polypropylene mesh packets. Theamounts of various reagents and solvent are used based upon the totalamount of active hydrogen present. An exemplary synthesis for a singleperalkylated peptide is discussed below.

This synthesis is for the permethylated peptide PerA-AGGFL, whoseN-terminal α-amine became a quaternary trimethylammonium group and whoseC-terminal carboxyl became an N-methyl-carboxamide. Thus, 0.60 g of 60percent NaH/oil about 25 milliequivalents (meq)! were added to 100 ml ofDMSO in a 250 ml polypropylene bottle with a ventilated screw cap toprovide a final concentration of 0.25M of NaH. After shaking for about30 minutes for the NaH to dissolve, 500 mg of the precursorpeptide-coupled resin (about 2.5 meq of active hydrogen) within apolypropylene mesh packet were added to the bottle.

The bottle and its contents were shaken on a reciprocal shaker for 14hours. At that time, 4.66 ml of CH₃ I (about 30 meq) were added directlyto the bottle and its reaction mixture. The resulting reaction mixturewas shaken for another 15 minutes.

The solution was poured off, and the packet and its contents were washedwith aliquots of 100 ml of each of DMSO, DMF, isopropanol, twice withdichloromethane and then methanol. The thus peralkylated resin-coupledpeptide was then dried under high vacuum and the peralkylated peptidecleaved using the standard high HF procedure discussed before.

Results for a similar peralkylation of this peptide-coupled resin arediscussed in Example 4 along with results for nineteen similarperalkylations.

EXAMPLE 2 Chemical Mixture Synthesis

These syntheses using 18 of the 20 naturally occurring amino acidderivatives (Cys and Trp omitted) are carried out substantially asdescribed in U.S. Pat. No. 4,631,211 and Example 1.

A cross-linked polystyrene resin is used as solid support that alsoincluded 0.93 milliequivalents (meq) of benzhydrylamine groups per gram.The solid support resin is typically utilized in an amount of 300milligrams (mg) so that 2.79 meq of resin-amine are initially providedin each reaction.

The mixture of amino acid derivatives noted in Table 2, below, at 0.5Min 4 ml of dimethylformamide (DMF) is used for each coupling, as about a7-fold molar excess over the amount of amine present, as resin-amine orafter deprotection to provide N-terminal amine (free amine) groups. Oneequivalent of DIPCDI as coupling agent and one equivalent ofN-hydroxylbenztriazole-H₂ O are used per equivalent of mixed amino acidderivative, so both are also present in about a 7-fold excess over thefree amine groups present.

                  TABLE 2.sup.1                                                   ______________________________________                                        Amino Acid     Weight.sup.2                                                   ______________________________________                                        Ala            19 mg                                                          Asp(Bn)        33 mg                                                          Glu(Bn)        36 mg                                                          Phe            20 mg                                                          Gly            15 mg                                                          His(DNP)       50 mg                                                          Ile            123 mg                                                         Lys(Cl--CBZ)   76 mg                                                          Leu            36 mg                                                          Met            18 mg                                                          Asn            37 mg                                                          Pro            27 mg                                                          Gln            39 mg                                                          Arg(Tsl)       82 mg                                                          Ser(Bn)        24 mg                                                          Thr(Bn)        44 mg                                                          Val            72 mg                                                          Tyr(Br--CBZ)   60 mg                                                          ______________________________________                                         .sup.1 Parenthesized designations in the left column are used by each         unless another parenthesized protecting group is shown. Bn = benzyl; DNP      dinitrophenyl; Tsl = toluenesulfonyl; CBZ = benzyloxy carbonyl; Cl--CBZ =     o-chlorobenzyloxy carbonyl; Br--CBZ = o-bromobenzyloxy carbonyl.              .sup.2 Milligrams (mg) of each protected amino acid derivative present in     a chemical mixture per 1 milliequivalent of resin --NH.sub.2 group.           Diisopropylcarbodiimide (DIPCD) used as coupling agent.                  

Each coupling is carried out at room temperature until there is noremaining free amine groups as in Example 1; about one hour.Deprotection and neutralizations are also carried out as in Example 1.

Each position of the precursor oligopeptide containing equimolar amountsof amino acid residues is added as described above. Using a 6-mer whosefifth position is occupied by one of eighteen predetermined amino acidside chains as exemplary, the above coupling provides a support-coupledone-mer peptide product of the formula X--B.

That support-coupled product is then divided into at least 18 aliquotsof equal weight, small portions of the preparation often being retainedfor analytical purposes. Those aliquots are enclosed in labeled porouspackets, as discussed in Example 1, and the 18 individual amino acidderivatives are reacted separately with those aliquots afterdeprotection and neutralization to form 18 support-coupled products ofthe formula O₅ X--B.

Those 18 labeled porous packets containing the O₅ X--B support-coupledproduct are then deprotected and neutralized together, and thoseproducts are together reacted again as discussed before with the mixedamino acid derivatives, while being maintained in their packets, to form18 sets of support-coupled products of the formula XO₅ X--B. Thisprocedure is repeated three more times to form the 18 support-coupled6-mer sets whose fifth position from the N-terminus is occupied by eachof the 18 different predetermined amino acid residues and whose otherpositions are occupied by equimolar amounts of the 18 amino acidresidues present in the reaction mixtures.

Where N-terminal acetyl groups are to be used, the N-terminal t-BOCgroups are removed, the resulting free amines neutralized and thesupport-coupled 6-mers are reacted with acetic anhydride to formN-acetyl (Ac) groups. The N-acetyl coupled peptides are thenperalkylated and cleaved from the solid support to form a plurality (18)of peralkylated 6-mer oligopeptide sets.

The above procedures are similarly used, as appropriate, to prepare theremaining five libraries of 18 sets (another 90 sets) having one ofeighteen predetermined peralkylated amino acid residues at predeterminedpositions 1-4 and 6, and mixtures of equal molar amounts of the 18peralkylated amino acid residues at the other peralkylated oligopeptidechain positions.

The relative equimolarity of coupling using the above procedure ascompared to the physical mixture methods in precursor oligopeptide setswas determined by amino acid analysis of support-coupled products from asingle coupling reaction. A commercial amino acid analyzer was utilizedfor these assays. The specific manipulations utilized are discussedhereinafter.

As is well known, even commercially available amino acid analyzers donot provide precise determinations because of several factors includingdecomposition of the amino acids, and the various reactions andresponses the machines must carry out and make. On the other hand, thephysical mixture method provides equimolar mixtures to a precision thatis much greater than that obtained by the machine alone.

Thus, a physical mixture process solid support-coupled product (X--B) ofone coupling reaction was prepared as in Example 1, deprotected, cleavedfrom the solid support resin and collected. A similar X--B solidsupport-coupled product was prepared by the chemical mixture method ofthis example. That X--B product was similarly deprotected, cleaved fromthe solid support resin and collected. Those samples were then sentamino acid analysis.

More specifically, after each of the above t-BOC, side chain-protectedmixtures was prepared, the t-BOC groups were removed, and the sidechains deprotected. Each of the two mixed amino acid-coupled solidsupports (X--B) was dried, and 20 mg of each resin-linked product wasplaced into 5 ml glass ampules. One milliliter of propionic acid:HCl(50:50, V/V) was added to each ampule. Air was removed from the ampuleswith a vacuum pump with care being taken not to aspirate the contents ofthe ampules. Each ampule was then sealed using a propane flame, whileunder vacuum. The sealed ampules were placed in a dry block heater andmaintained at 130° C. for two hours to cleave the reacted amino acidsfrom the solid support resin and form hydrolyzate solutions.

Thereafter, upon cooling to room temperature, the ampules were brokenopen and their contents filtered into separate 12-75 mm culture tubes.Aliquots (20 μl) of the hydrolyzate solution were placed into 5-50 mmculture tubes in duplicate. Those samples were coded, dried and sealed.

The sealed, coded samples were sent to Core Laboratories, New Orleans,La. for amino acid analysis. The results of that analysis are shownbelow, for each sample. In addition, because it is known that thephysical mixture method provides more precise results than does aminoacid analysis, the percentage of deviation from equimolarity for thechemical mixture method was determined by presuming that the valueobtained for the individual amino acid residues obtained from thephysical mixture method was the correct value of one-eighteenth molepercent (5.56 percent). It is noted that Glu and Gln analyze together asdo Asp and Asn because the resin-cleaving step also destroys the Gln andAsn amide bonds, forming Glu and Asp, respectively.

    ______________________________________                                               Mole Percent   Deviation from                                          Amino    Physical    Chemical Equimolarity                                    Acid     Mixture     Mixture  (Percent)                                       ______________________________________                                        Asp,Asn  13.84       16.73    +21                                             Glu,Gln  10.87       11.99    +10                                             Ser      4.11        4.14     -1                                              Gly      5.13        5.04     -2                                              His      4.84        3.16     -35                                             Arg      6.57        5.03     -23                                             Thr      5.48        6.10     +11                                             Ala      6.36        6.48     +2                                              Pro      7.22        7.28     +1                                              Tyr      4.31        3.53     -18                                             Val      6.08        6.76     +11                                             Met      3.38        4.13     +22                                             Ile      5.08        4.07     -20                                             Leu      6.58        5.95     -10                                             Phe      5.17        3.78     -27                                             Lys      4.96        5.85     +18                                             ______________________________________                                    

EXAMPLE 3 Synthesis of Peptide Mixtures on Cotton Carriers

Twenty discs cut out of commercially available cotton fabric (diameter4.7 cm) are shaken for 15 minutes in 50 ml of dichloromethane (DCM)containing 25 percent trifluoroacetic acid (TFA). The discs are thentaken out and placed into a flat ceramic funnel with the same diameteras the cotton discs. The funnel is placed on top of an 1000 ml suctionflask with an outlet to a vacuum pump. The 25 percent TFA/DCM is removedfrom the cotton discs into the suction flask under reduced pressure. Thecotton discs are then washed with DCM (2×10 ml), DCM containing 5percent DIEA (2×10 ml) and DCM (2×10 ml) again. The washings are done byadding the wash solution to the funnel holding the cotton discs andremoval of the solvent with a vacuum pump. After the last wash thecotton discs are removed and air dried. All manipulations are at roomtemperature unless otherwise stated.

A. Manual Synthesis

Fmoc-Glycine (1.118 g, 4 mmol), N-hydroxybenztriazole (HOBt) (540 mg, 4mmol), N-methylimidazole (NMI) (656 μl, 8 mmol) and DIPCDI (626 μ, 4mmol) are dissolved in 6.7 ml DMF. This corresponds to a 0.5 MFmoc-Gly/HOBt/DIPCDI, 1M NMI solution. The cotton discs are soaked withthis solution in a 20 ml scintillation vial and maintained for threehours. After transferring the discs to the ceramic funnel, the cottoncarriers are washed with DMF (3×10 ml) and DCM (2×10 ml) as describedabove. This procedure is repeated once more identically.

The general peptide mixture and single, predetermined peptide couplingprocedure is as follows:

1. Fmoc-deprotection: 20 percent piperidine/DMF, 15 minutes.

2. Wash: 3×DMF, 3×DCM.

3. Coupling: 0.3M Fmoc-amino acid/HOBt/DIC in DMF, 90 minutes - twohours.

4. Wash: 3×DMF, 2×DCM.

More specifically, the twenty cotton discs, placed into the ceramicfunnel, are soaked with 10 ml 20 percent piperidine/DMF, and maintainedfor 15 minutes. After removing the 20 percent piperidine/DMF, the cottondiscs are washed with DMF (3×10 ml) and DCM (2×10 ml) as describedabove.

(a) Coupling of the same amino acid to all cotton discs

The Fmoc-amino acid to be coupled (2.4 mmol), HOBt (324 mg, 2.4 mmol)and DIPCDI (380 μl, 2.4 mmol) are dissolved in 7.6 ml DMF. Thiscorresponds to a 0.3 M Fmoc-amino acid/HOBt/DIC solution. The cottondiscs are soaked with this solution in a 20 ml scintillation vial andmaintained for 90 minutes. After transferring the discs to the ceramicfunnel, the coupling solution is removed, and the cotton carriers arewashed with DMF and DCM, as before.

(b) Coupling of another amino acid to each cotton disc (O-coupling)

The 20 natural amino acids (0.12 mmol each) are separately dissolved in0.4 ml of a 0.3M solution of HOBt and DIPCDI in DMF (324 mg HOBt and 380μl DIPCDI dissolved in 7.6 ml DMF). The cotton discs are labeled as toamino acid identify with the letters A through Y, soaked with the aminoacid solution, labeled with the letter of the amino acid of thesolution, and maintained for 90 minutes. After transferring the discs tothe ceramic funnel, the cotton discs are washed with DMF and DCM, asbefore.

(c) Coupling of the amino acid mixture (X-coupling)

A 0.3M solution of the 20 natural amino acids except Cys in the molarratio of Table 2 and HOBt in DMF is prepared and aliquoted. The aliquots(7.6 ml each) are stored at -20° C. Before the coupling, the mixturealiquot is warmed up to room temperature, followed by addition of 380 μlDIPCDI. After 20 minutes (preactivation), the 19 cotton discs are soakedwith this solution and maintained for two hours. After transferring themto the ceramic funnel, the cotton discs are washed with DMF and DCM, asbefore.

After coupling of the last (N-terminal) amino acid or mixture, thecotton discs are Fmoc-deprotected and washed. The deprotected cottondiscs are soaked with 8 ml of a mixture of acetic anhydride/pyridine/DMF1:2:3 (v/v/v) and maintained for 60 minutes. After transferring them tothe ceramic funnel, the cotton discs are washed with DMF and DCM.

The acetylated cotton discs are placed into a bottle containing 30 ml 50percent TFA, 5 percent triisobutylsilane in DCM and maintained for twohours. After pouring off the solution, 100 ml DCM are added and thebottle shaken for two minutes. This wash is repeated twice with DCM,then three times with 5 percent DIEA/DCM and again three times with DCM.The cotton discs are taken out, blotted between layers of filter paperand air dried. The dry cotton discs are cut into small discs (diameter 7mm) with an ordinary hole puncher, labeled and refrigerated.

Peralkylation is then carried out as discussed before.

B. Machine Synthesis

The synthesis is done as described in allowed U.S. Pat. No. 5,202,418 toLebl et al., whose disclosures are incorporated by reference, and above.The essential difference between the manual synthesis of peptidemixtures and the synthesis of individual peptides on the synthesizermachine is the following: The manually prepared mixtures are synthesizeddirectly on the glycine-cotton. Upon alkaline hydrolysis of theglycine-cotton ester, the cotton-cleaved peptides therefore contain anadditional C-terminal Gly residue. In case of the synthesis ofindividual peptides on the machine synthesizer, a TFA-cleavable linker,in this case N-f-Moc-2,4-dimethoxy-4'-(carboxymethyloxy)-benzhydrylaminefor the synthesis of peptide amides, is coupled onto the amino group ofthe glycine-cotton ester. After Fmoc-deprotection of the linker, thefirst amino acid of the peptide is coupled to the amino group of thelinker. Upon cleavage of the peptides with TFA, simultaneously with thedeprotection of side chains, the set of oligopeptide amides is formed,with the linker and the glycine remaining bound to the cotton.Peralkylation of the cleaved sets is then carried out. Peralkylation canalso be carried out while the precursor peptide is bound to the solidsupport.

EXAMPLE 4 Pentamer Permethylation

Twenty model pentameric solid support-bound oligopeptides were preparedas described in U.S. Pat. No. 4,631,211. The C-terminal four positionswere identical for each, with the N-terminal residue being varied amongthe twenty naturally occurring amino acid residues. The support-boundprecursor pentamers were peralkylated as discussed before.

Briefly, the support-bound pentamers (50 mg each), each in their ownpolypropylene mesh containers, were shaken for 16 hours at roomtemperature in a DMSO solution of 0.25 molar sodium hydride (24 ml, 8meq.). Methyl iodide (neat 1.1 ml, 24 meq.)was then added to thesolution at an 4-fold excess over the moles of base. The peralkylationreaction was permitted to continue for 15 minutes or until the reactionmixture came to room temperature. Following washes with DMF (3×5 ml),isopropanol (IPA; 2×5 ml), DCM (3×5 ml) and methanol (1×5 ml), theperalkylated support-bound peptides were dried under high vacuum. Theperalkylated oligopeptides were cleaved from the support resin using 7.5percent anisole in HF (5 ml) for one and one-half hours at zero degreesC, dried under a high vacuum, extracted with 10 ml of water andlyophilized. Use of 10 volume percent acetic acid in water for theextraction provides an enhanced result with more hydrophobic alkylgroups.

The individual peralkylated oligopeptides were recovered and subjectedto reversed phase high performance liquid chromatograph (RP-HPLC) andidentified by laser-desorption mass spectral analysis (Kratos).Analytical RP-HPLC was performed on a Bydac C₁₈ column (Hesperia,Calif.) using a Beekman System Gold™ HPLC system monitored at 215 nm;Solvents A, 0.05 percent TFA/H₂ O; Solvent B, 0.05 percent TFA/CH₃ CN;gradient, 5-65 percent B. Preparative RP-HPLC was performed on a WatersC₁₈ column (5 cm×25 cm) on a Waters Delta Prep™ HPLC System monitored at215 nm; Solvents A and B as above. The results of those analyses areshown in Table 3, below, using single letter code of unalkylated aminoacids for the peralkylated (permethylated) residue.

                  TABLE 3                                                         ______________________________________                                        Model Peptide Side Chain Modifications                                                         Post                                                                          Methylation                                                                             Added    HPLC                                      Peralkylated     Molecular Number of                                                                              Purity                                    Sequence.sup.1   Weight    Methyls  (Percent).sup.3                           ______________________________________                                        PerA-AGGFL (SEQ ID NO: 1)                                                                      575.3     8        91                                        PerA-CGGFL (SEQ ID NO: 2)                                                                      620.8     9        50                                        PerA-DGGFL (SEQ ID NO: 3)                                                                      633.6     9        60                                        PerA-EGGFL (SEQ ID NO: 4)                                                                      647.8     9        75                                        PerA-FGGFL (SEQ ID NO: 5)                                                                      650.9     8        91                                        PerA-GGGFL (SEQ ID NO: 6)                                                                      661.0     8        92                                        PerA-HGGFL (SEQ ID NO: 7)                                                                      655.8     9        40                                        PerA-IGGFL (SEQ ID NO: 8)                                                                      617.2     8        90                                        PerA-KGGFL (SEQ ID NO: 9)                                                                      673.0     11       30                                        PerA-LGGFL (SEQ ID NO: 10)                                                                     617.2     8        81                                        PerA-MGGFL.sup.2 (SEQ ID NO: 11)                                                               651.8     8        70                                        PerA-NGGFL (SEQ ID NO: 12)                                                                     649.9     10       86                                        PerA-PGGFL (SEQ ID NO: 13)                                                                     588.0     7        90                                        PerA-QGGFL (SEQ ID NO: 14)                                                                     660.0     10       80                                        PerA-RGGFL (SEQ ID NO: 15)                                                                     547.8     11       75                                        PerA-SGGFL (SEQ ID NO: 16)                                                                     591.7     8        95                                        PerA-TGGFL (SEQ ID NO: 17)                                                                     604.9     8        95                                        PerA-VGGFL (SEQ ID NO: 18)                                                                     603.2     8        95                                        PerA-WGGFL (SEQ ID NO: 19)                                                                     704.8     9        70                                        PerA-YGGFL (SEQ ID NO: 20)                                                                     681.5     9        81                                        ______________________________________                                         .sup.1 Each peralkylated oligopeptide contained a trimethylammonium           Nterminal nitrogen atom, a methyl group on each amido nitrogen of the         backbone, appropriately methylated side chains and a Cterminal                Nmethylcarboxamide group that are not shown in the peralkylated sequences     above.                                                                        .sup.2 The methionine was present as methionine sulfoxide.                    .sup.3 Purities of the crude compounds were determined by analytical          RPHPLC.                                                                  

EXAMPLE 5 Binding to Opioid Receptors

The enkephalins were the first natural ligands found for the opioidreceptors. These molecules bind to three known receptor subclasses (mu,delta and kappa) with differing affinities reviewed in Schiller,Progress in Medicinal Chemistry, Ellis et al. eds., Elsevier SciencePublishers, U.K. (1990) pages 301-340!. The competitive binding studiesherein utilized a radiolabeled analog of met-enkephalin, ³ H! -D-Ala²,MePhe⁴,Gly-Ol⁵ !enkephalin (DAGO) that is known to bindspecifically to the mu receptor in competitive binding studies withpositional libraries of peralkylated hexamer sets; i.e., peralkylated5×sets.

Two libraries of libraries of sets were used for these assays. The firstcontained an N-terminal pyroglutamoyl group (from pyrrolidone carboxylicacid), and the second library contained an a-quaternarytrimethylammonium group. Both libraries were permethylated and containedeach of the 20 natural amino acids with Met present as the sulfoxide andN-methyl caboxamido C-termini. These libraries were prepared asdiscussed in Example 1 and were permethylated as discussed before.

A. N-Pyroglutamoyl-Terminated Libraries

Scanning the first or N-terminal position (the 1-position) showed thatpermethylated Asp, Glu, Gly, Lys, Met, Asn, Pro, Gln, Ser, Thr and Valprovided the greatest inhibition, between 50 and 60 percents, withpermethylated Thr and Glu providing the greatest inhibition.

Scanning the 2-position showed that permethylated Asp, Glu, Gly, Ile,Asn, Pro, Gln and Thr provided the greatest inhibition, between 50 and60 percents, with permethylated Asn being best.

Scanning the 3-position showed that only permethylated Glu provided aninhibition greater than 60 percent, with nine other residuespermethylated Ala, Asp, Gly, Met, Asn, Pro, Gln, Ser and Thr providinginhibitions between 50 and 60 percents.

The 4-position scan showed that permethylated Asp, Glu and Met providedabout 60 percent binding inhibition and eight more permethylatedresidues exhibition inhibitions between 50 and 60 percents; i.e., Ala,Gly, Asn, Pro, Gln, Ser, Thr and Val.

The 5-position scan showed permethylated Glu to provide an inhibition ofmore than 60 percent, with permethylated Met, Ser and Thr exhibiting analmost 60 percent inhibition.

Scanning of the 6-position, the C-terminus, showed that permethylatedGlu provided almost 100 percent inhibition of binding, with no otherpermethylated residue providing more than 70 percent inhibition.

Permethylated His exhibited an inhibition of 20 percent or less at eachposition. Permethylated Trp, Phe and Arg were also uniformly poorinhibitors at each scanned position.

The enkephalins and DAGO both contain two aromatic residues. The aboveresults thus indicate that peralkylated aromatic residues are notimportant to binding here, thus opening the way to new relativelyhydrolytically stable enkephalin inhibitors.

B. Trimethylammonium-Terminated Libraries

The same precursor support-coupled peptides used in A, above, butlacking the N-pyroglutamoyl group were permethylated as discussed beforeand cleaved from the resin support to provide six libraries of 20library sets of 5×oligopeptides. The N-terminal residues here hadα-trimethylammonium groups.

Scanning of the 1-position showed that only permethylated Glu exhibiteda binding inhibition of greater than 40 percent. Permethylated Met, Pro,Gln and Ser exhibited inhibitions of between 30 and 40 percent.

Scanning the 2-position again showed permethylated Glu to be alone atmore than 40 percent inhibition. Permethylated Asp, Gly, Met, Asn, Pro,Gln and Thr exhibited inhibitions of between about 30 and 40 percents.

Position 3 again showed permethylated Glu to provide the best inhibitionat between about 30 and 35 percents, with permethylated Met and Gln alsoexhibiting greater than about 30 percent inhibition.

Permethylated Met provided the best inhibition at position 4, followedby permethylated Asn, Glu, Gln and Gly. Each inhibition was about 30percent or greater, but less than 40 percent.

The scan of the 5-position showed that only permethylated Gln exhibitedinhibition greater than 40 percent. Permethylated Ala, Asp, Met, Asn,Ser and Thr exhibited inhibitions between 30 and 40 percent.

The scan of the 6-position showed permethylated Gly, Met and Thr toexhibit inhibitions of between about 40 percent and about 50 percent.

Permethylated His, the aromatics, Cys, Arg and Lys all exhibited poorbinding inhibition at each position in these assays.

Binding inhibitions exhibited by these libraries were generally lowerthan those illustrated before. These libraries exhibited somewhatgreater differences in binding inhibitions.

The above assays were carried out using opioid receptors from rat brainsprepared as follows. Particulate membranes were prepared using amodification of the method described by Pasternak et al., Mol. Pharm.,11:340-351 (1975). Rat brains frozen in liquid nitrogen were obtainedfrom Rockland Inc. (Gilbertsville, Pa.). The brains were thawed, thecerebella were removed, and the remaining tissue was weighed. Each brainwas individually homogenized in 40ml Tris-HCl buffer (5OmM, pH 7.4, 4°C.) and centrifuged (Sorvall RC5C SA-600 16000 rpm) for ten minutes. Thepellets were resuspended in fresh Tris-HCl Buffer and incubated at 37°C. for 40 minutes. Following incubation, the suspensions werecentrifuged as before, the resulting pellets were resuspended in 100volumes of Tris buffer, and the suspensions combined. Membranesuspensions were prepared and used in the same day. Protein content ofthe crude homogenates ranged from 0.15-0.2 mg/ml as determined using themethod described by Bradford, Anal. Biochem., 72:248-254 (1976).

Binding assays were carried out in polypropylene tubes. Each tubecontained 0.5 ml of membrane suspension, 8 nM of ³ H!-D-Ala²,MePhe⁴,Gly-Ol⁵ !enkephalin (DAGO) (specific activity=36 Ci/mmole,160,000 cpm/tube; obtained from Multiple Peptide Systems, Inc., SanDiego, Calif. through NIDA drug distribution program 271-90-7302), and 2mg/ml of permethylated peptide mixture and Tris-HCl buffer in a totalvolume of 0.65 ml. Assay tubes were incubated for 60 minutes at 25° C.The reaction was terminated by filtration through GF-B filters on aTomtec harvester (Orange, Conn.). The filters were subsequently washedwith 6 ml of Tris-HCl buffer, 4° C. Bound radioactivity was counted onan LKB Beta-plate Liquid Scintillation Counter and expressed in countsper minute (cpm). To determine inter- and intra-assay variation,standard curves in which ³ H!-DAGO was incubated in the presence of arange of concentrations of unlabeled DAGO (0.13-3900 nM) were includedin each plate of each assay (a 96-well format was used).

EXAMPLE 6 Antimicrobial Activity Against S. aureus

The two types of libraries of positional libraries of Example 5 werealso scanned against Staphylococcus aureus a Gram-positive bacterium.Concentrations of those 5×permethylated peptide mixtures that inhibitedcellular growth by 50 percent (IC₅₀) values were determined.

Residues of each library type and set member are provided below whoseIC₅₀ values were within a factor of about two of the most potentresidues. IC₅₀ values are in mg/ml.

    ______________________________________                                        A. N-Pyroglutamoyl-Terminated Libraries.sup.1                                 ______________________________________                                        Position 1            Position 2                                              Residue Average IC.sub.50                                                                           Residue Average IC.sub.50                               ______________________________________                                        Tyr     500.57        Tyr     597.67                                          Trp     792.61        Ile     781.86                                          Ile     871.18        Trp     995.69                                                                Leu     1031.02                                         ______________________________________                                        Position 3            Position 4                                              Residue Average IC.sub.50                                                                           Residue Average IC.sub.50                               ______________________________________                                        His     394.85        His     321.62.sup.2                                    Tyr     519.62        Tyr     538.86                                          Trp     639.02        Leu     640.90                                          Val     797.33                                                                ______________________________________                                        Position 5        Position 6                                                  Residue Average IC.sub.50                                                                           Residue Average IC.sub.50                               ______________________________________                                        His     217.222       His     152.41.sup.2                                    ______________________________________                                         .sup.1 Starting concentrations of 2500 μg/ml of permethylated sets wer     used. Each IC.sub.50 value is an average of two assay results.                .sup.2 Only one assay was conducted.                                     

The above results indicate that the permethylated aromatic residues Tyrand Trp that were unimportant in the assays of Example 5 are quiteimportant near the N-terminus in these assays. Similarly, His, which wasalways the worst residue in inhibiting binding in Example 5 is importantnear or at the C-terminus. Similarly, permethylated Asp, Glu, Asn, Gin,Ser, Thr and Gly that were important in the assays of Example 5typically provided the highest IC₅₀ values.

    ______________________________________                                        B. N-Trimethylammonium-Terminated Libraries.sup.1                             Position 1            Position 2                                              Residue Average IC.sub.50                                                                           Residue Average IC.sub.50                               ______________________________________                                        Tyr     94.41         Phe     93.96                                           Trp     103.35        Tyr     131.54                                          Phe     115.49        Ile     141.56                                          Ile     138.92        Trp     167.46                                          His     157.67        His     192.85                                          Leu     188.85                                                                ______________________________________                                        Position 3            Position 4                                              Residue Average IC.sub.50                                                                           Residue Average IC.sub.50                               ______________________________________                                        Phe     89.93         Trp     93.29                                           Ile     93.01         Phe     93.50                                           Trp     100.14        Ile     108.05                                          Leu     135.07        His     121.91                                          Tyr     136.15        Tyr     161.67                                          Cys     146.89        Leu     183.48                                          Val     187.18                                                                ______________________________________                                        Position 5            Position 6                                              Residue Average IC.sub.50                                                                           Residue Average IC.sub.50                               ______________________________________                                        Phe     95.72         His     91.37                                           His     101.57        Phe     93.77                                           Trp     121.21        Trp     93.78                                           Ile     126.18        Tyr     145.18                                          Tyr     156.50        Ile     154.41                                          Leu     171.34                                                                ______________________________________                                        Positive Controls                                                             Drug           Average IC.sub.50                                              ______________________________________                                        Oxacillin      0.106                                                          Erythromycin   0.184                                                          ______________________________________                                         .sup.1 Starting concentrations of 2500 μg/ml for the less active           permethylated sets were used, with starting concentrations of 1250            μg/ml for the more active sets. IC.sub.50 Values are an average of two     assay results.                                                           

The most active of these latter, N-trimethylammonium-terminated, setswere about two- to about five-fold more active than the prior sets ininhibiting growth of this bacterium. These latter sets also showed animportance of permethylated aromatic residues, particularlyphenylalanine (Phe), near the N-terminus that continued through eachposition. Histidine was also important near the C-terminus of thesesets, but in a less clear-cut way than noted in the first sets assayed.The same residues found important in Example 5 but unimportant againstS. aureus in the first assays of this example were also foundunimportant here. Methionine sulfoxide that was also important inExample 5 was relatively unimportant here.

EXAMPLE 7 Permethylated Oligo-Phe Peptides

In view of the apparent importance of permethylated Phe residues at eachposition in the scans of Example 6, a series of permethylated homoPheC-terminal N-methylamide peptides was prepared, as was permethylated PheN-methylamide itself. Each permethylated peptide and the permethylatedamino acid contained an N-terminal trimethylammonium group and aC-terminal N-methylamide group.

It was thought that the observed preference for peralkylated Pheresidues may have been due to a real preference for one or two suchresidues that was evidenced as a frame shift in the positional scans.Thus, an individual peralkylated amino acid and individual peralkylatedpeptides, rather than sets or libraries of sets were prepared andassayed as illustrative and to examine the possibility of the resultsbeing due to a frame shift.

The permethylated amino acid and peptides were screened against S.aureus and Streptoccus sanguis, as described below, and IC₅₀ and MIC(minimum inhibitory concentration; minimum concentration needed toinhibit about 100 percent bacterial growth) values were determined.Average results from those studies are shown below in Tables 4 and 5,along with results for the antibiotics oxacillin and erythromycin andN-acetyl hexaPhe amide (N-Ac-hexaPhe) as controls in the S. aureusstudy.

                  TABLE 4                                                         ______________________________________                                        Assays Against S. aureus                                                      Peralkylated        IC.sub.50 Value                                                                         MIC Value                                       Sequence.sup.1      (μM)   (μM)                                         ______________________________________                                        PerA-F                  >500      >500                                        PerA-FF                 >500      >500                                        PerA-FFF                288       >500                                        PerA-FFFF  SEQ ID NO: 21                                                                              116       250-500                                     PerA-FFFFF SEQ ID NO: 22                                                                              19        21-31                                       PerA-FFFFFF                                                                              SEQ ID NO: 23                                                                              7         11-15                                       PerA-FFFFFFF                                                                             SEQ ID NO: 24                                                                              2.5       3-4                                         PerA-FFFFFFFF                                                                            SEQ ID NO: 25                                                                              5         6-8                                         Controls                                                                      N--Ac-hexaPhe       >500      >500                                            Oxacillin           0.042     0.125                                           Erythromycin        0.184     0.5                                             ______________________________________                                         .sup.1 Each peralkylated oligopeptide contained a trimethylammonium           Nterminal nitrogen atom, a methyl group on each amido nitrogen of the         backbone, appropriately methylated side chains and a Cterminal                Nmethylcarboxamide group that are not shown in the peralkylated sequences     above.                                                                   

                  TABLE 5                                                         ______________________________________                                        Assays Against S. sanguis                                                     Peralkylated        IC.sub.50 Value                                                                         MIC Value                                       Sequence.sup.1      (μM)   (μM)                                         ______________________________________                                        PerA-F                  >500      >500                                        PerA-FF                 >500      >500                                        PerA-FFF                446       >500                                        PerA-FFFF  SEQ ID NO: 21                                                                              85        125-250                                     PerA-FFFFF SEQ ID NO: 22                                                                              20        25-30                                       PerA-FFFFFF                                                                              SEQ ID NO: 23                                                                              5         7-8                                         PerA-FFFFFFF                                                                             SEQ ID NO: 24                                                                              2.3       3-4                                         PerA-FFFFFFFF                                                                            SEQ ID NO: 25                                                                              7         8-9                                         ______________________________________                                         .sup.1 Each peralkylated oligopeptide contained a trimethylammonium           Nterminal nitrogen atom, a methyl group on each amido nitrogen of the         backbone, appropriately methylated side chains and a Cterminal                Nmethylcarboxamide group that are not shown in the peralkylated sequences     above.                                                                   

As can be seen from the above data, the positional scanning processprovided a basis for obtaining a permethylated oligopeptide whosepotency was less, but similar to those of recognized antibiotics. Theabove results also indicate that the scanning results were correct andnot due to a frame shift. These results were obtained using anunoptomized sequence in that Phe was not the optimal residue at eachposition in the positional scans of Example 6, but was near optimal anduse of a homoPhe permethylated heptapeptide was convenient forillustration. Even though the homoPhe permethylated heptapeptide wasused illustratively, the potency of the permethylated peptide was shownto increase as each residue was added, with an overall potency increaseof over 2 orders of magnitude being shown.

Staphylococcus aureus (ATCC 29213) were grown overnight (about 18 hours)at 37° C. in cation-adjusted Mueller-Hinton (CAMH) broth. This culturewas reinoculated and incubated at 37° C. to reach the exponential phaseof bacterial growth; i.e., a final bacterial suspension containing about10⁵ to 5×10⁵ colony-forming units (CFU)/ml. The concentration of cellswas established by plating 100 μl of different dilutions of the culturesolution (e.g., 10⁻², 10⁻³ and 10⁻⁴) onto solid agar plates. Followingan overnight (about 18 hours) incubation at 37° C., the CFU thus formedwere counted on each agar plate.

96-Well tissue culture plates were utilized, with eight wells per platecontaining only medium as control blanks, whereas eight other wellscontained medium plus cells as a positive growth control. These controlswere used to detect possible medium contamination and to provide ameasure of uninhibited growth of the microorganisms.

For determination of IC₅₀ values (concentrations necessary to inhibit 50percent growth of bacteria), peralkylated oligopeptide sets were addedto the bacterial suspension at concentrations noted before. The plateswere incubated overnight (about 18 hours) at 37° C., and the opticaldensity (OD) determined at 620 nm after different times of incubation.IC₅₀ Values were then determined from the OD data.

EXAMPLE 8 Single Permethylated Oligopeptides

Based on the results shown in Example 6 against S. aureus using the 120scanning N-trimethylammonium-terminated permethylated C-terminalmethylamido oligopeptide sets, individual peptides were synthesizedrepresenting all combinations of the six amino acids chosen for thefirst position (Trp, Phe, Tyr, His, Ile and Leu); Phe for the secondposition; Trp, Ile and Phe for the third position; Trp and Phe for thefourth position; and Phe and His for both the fifth and sixth positions.Following their synthesis, the resultant 144 individual, permethylatedpeptides (6×1×3×2×2×2) were assayed for their antimicrobial activity. Ofthe 144 peptides prepared, 41 had IC_(50's) <50 μg/ml, while 51 hadIC_(50's) ≧250 μg/ml against S. aureus. Table 6 lists the 10 most activesequences and data from their use.

                                      TABLE 6                                     __________________________________________________________________________    Antimicrobial and Hemolytic Activities of Permethylated Peptides.sup.1                       S. aureus                                                                            MRSA   S. sanguis                                                                           % hemolysis                               μg/ml                                                                              Sequence.sup.2                                                                       IC.sub.50                                                                        MIC IC.sub.50                                                                        MIC IC.sub.50                                                                        MIC at 350                                    __________________________________________________________________________    SEQ ID NO: 26                                                                         PerM-LFIFFF                                                                          6  11-15                                                                             7   8-10                                                                             1.7                                                                               3-5                                                                              12                                        SEQ ID NO: 27                                                                         PerM-FFIFFF                                                                          6  11-15                                                                             7   8-10                                                                             14 20-40                                                                             0                                         SEQ ID NO: 23                                                                         PerM-FFFFFF                                                                          7  11-15                                                                             7   8-10                                                                             9  15-20                                                                             16                                        SEQ ID NO: 28                                                                         PerM-LFFFFF                                                                          10 21-31                                                                             8   9-10                                                                             14 20-40                                                                             10                                        SEQ ID NO: 29                                                                         PerM-FFFFHF                                                                          11 15-21                                                                             18 21-42                                                                             19 30-40                                                                             3                                         SEQ ID NO: 30                                                                         PerM-LFIFFH                                                                          12 21-31                                                                             20 25-42                                                                             19 30-40                                                                             1                                         SEQ ID NO: 31                                                                         PerM-LFFFHF                                                                          12 21-31                                                                             13 15-21                                                                             19 30-40                                                                             4                                         SEQ ID NO: 32                                                                         PerM-LFIFHF                                                                          12 21-31                                                                             14 15-21                                                                             13 20-40                                                                             7                                         SEQ ID NO: 33                                                                         PerM-LFFFFH                                                                          13 21-42                                                                             16 21-42                                                                             22 30-40                                                                             3                                         SEQ ID NO: 34                                                                         PerM-FFIFFH                                                                          14 31-42                                                                             19 21-42                                                                             18 30-40                                                                             0                                         __________________________________________________________________________     .sup.1 All numerical data are in μg/ml.                                    .sup.2 Each peralkylated oligopeptide contained a trimethylammonium           Nterminal nitrogen atom, a methyl group on each amido nitrogen of the         backbone, appropriately methylated side chains and a Cterminal                Nmethylcarboxamide group that are not shown in the peralkylated sequences     above.                                                                   

The permethylated forms of FFIFFF-NH₂, FFFFFF-NH₂ and LFIFFF-NH₂ (SEQ IDNO's:27, 23 and 26) were found to exhibit the greatest activity of theseries, with each having an MIC lower than 15 μg/ml. The permethylatedhepta- and octa-Phe peptides (SEQ ID NO's:24 and 25) of Example 7exhibited similar activities against MRSA. In contrast, the quaternaryammonium salt of non-permethylated FFFFFF-NH₂ (SEQ ID NO:35) showed noactivity (IC₅₀ >250 μg/ml). Similar activities were found against MRSAand S. Sanquis (Table 6), whereas none of the permethylated peptides orpeptide mixtures exhibited activity against E. coli or C. albicans(IC_(50's) >600 μg/ml) . Also, none of the mixtures, nor any of theindividual permethylated peptides, had significant toxicity as evidenceby lysis of human red blood cells (RBC's) (Table 6). The activity ofthese new compounds therefore appears to be highly specific forGram-positive bacteria.

These compounds showed activities similar to a range of previouslydescribed peptides made up of L-amino acids Houghten et al., Nature,354:84-86 (1991); Houghten et al., Biotechniques, 13:412-421 (1992); andHoughten et al., in Innovation and Perspectives in Solid PhaseSynthesis, Epton ed., Solid Phase Conference Coordination, Andover, UK(1992), pp. 237-239!, and, in contrast to these L-amino acid peptides,they appear to be completely stable to proteolytic enzymes (as indicatedhereinafter). The physical-chemical properties of these compounds(enhanced resistance to proteolytic enzymes, excellent water solubility,favorable aqueous/organic partitioning characteristics) add value totheir potential as therapeutic leads. Surprisingly, only a small numberof the permethylated peptides having either Trp or Tyr in theirsequences were found to be active. In experience with the L-amino acidlibrary, it is consistently found that Trp- and Tyr-containing peptidesto have antimicrobial activity, which has not been the case when thepeptides are permethylated Houghten et al., Nature, 354:84-86 (1991);Houghten et al., Biotechniues, 13:412-421 (1992); and Houghten et al.,in Innovation and Perspectives in Solid Phase Synthesis, Epton ed.,Solid Phase Conference Coordination, Andover, UK (1992), pp. 237-239!.This is being investigated separately using the same iterative approachthat was used to identify the activity L-amino acid peptides.

Assays using S. aureus were carried out as described before.Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591,Streptococcus sanquis ATCC 10566, Escherichia coli ATCC 25922 andCandida albicans ATCC 10231 were used in the bioassays (ATCC, Rockville,Md.). To initiate the exponential phase of bacterial growth prior to theassay, a sample of bacteria grown overnight (about 18 hours) at 37° C.in cation-adjusted Mueller Hinton broth (CAMH, for E. coli and S.aureus--Becton Dickinson Microbiology Systems, Cockeysville, Md.), orBrain Heart Infusion broth (BHI, for S. sanguis), was reinoculated in a2×broth and incubated at 37° C. MRSA was grown at 35° C. incation-adjusted MH broth in a similar manner. Prior to the assay, twocolonies of newly grown C. albicans culture were inoculated in 5ml ofphosphate buffer saline (PBS-35mM phosphate buffer, 0.15M NaCl-pH 7.0),and diluted in yeast medium broth. The bacteria and yeast suspensionswere adjusted turbidimetrically so that a final concentration of 10⁵ to5×10⁵ colony-forming units (CFU)/ml was used in all assays.

The assays were carried out in 96-well tissue culture plates (Costar,Pleasanton, Calif.) as described in Blondelle et al., Biochemistry,31:12688-12694 (1992), incorporated herein by reference. In brief, thebacterial suspension was added to an equal volume of the mixture ofpermeythylated compounds diluted in H₂ O at concentrations derived fromserial two-fold dilutions varying from 2500 μg/ml to 1 μg/ml. The plateswere then incubated overnight at 37° C. The relative percent growthfound for each compound (or mixture of compounds) was then determined bythe optical density at 620 nm (OD₆₂₀) using a Titertek Multiskan Plusapparatus (Flow Laboratories, McLean, Va.). The concentration necessaryto inhibit 50 percent bacterial growth (IC₅₀) was then calculated usingthe software program GRAPHPAD (ISI, San Diego, Calif.). The minimuminhibitory concentrations (MIC's) were defined as the minimumconcentration of peptide at which there was no change in OD₆₂₀ betweentime 0 and 21 hours. In a similar manner, the plates containing C.albicans suspension were incubated for 48 hours at 30° C.

Hemolytic activities of the peptides were determined using human redblood cells (RBC's). The assays were carried out in 96-well tissueculture plates against a 0.25 percent RBC suspension as described inBlondelle et al., Biochim. Biophys. Acta, 1202:331-336 (1993),incorporated herein by reference. Briefly, individual permethylatedpeptides or peptide mixtures were added to the RBC solution atconcentrations derived from serial two-fold dilutions varying from 650to 64 μg/ml. Following a one hour incubation at 37° C., the plates werecentrifuged at 2800 rpm for 5 minutes. The supernatant was separatedfrom the pellet and its OD measured at 414 nm. Triton (1 percent) andPBS were used as 100 percent and 0 percent hemolysis controls,respectively.

EXAMPLE 9 Racemization Study

From the literature, is was anticipated that the conditions used herefor permethylation would result in minimal racemization or C.sub.α-methylation Challis et al., The Chemistry of Amides, Zabicky ed.,Interscience, New York (1970) pp. 731-857; Coggins et al., Can J. Chem.,49:1968-1971 (1971)!. Because the increased acidity of the C-hydrogen ofaromatic amino acids such as Phe Challis et al., The Chemistry ofAmides, Zabicky ed., Interscience, New York (1970) pp. 731-857; Cogginset al., Can J. Chem., 49:1968-1971 (1971)! makes them more prone toracemization and/or C.sub.α -methylation, a test series was devised inwhich the four possible stereoisomers of GlyGlyPheLeu-NH₂ (SEQ ID NO:36)were synthesized and analyzed by RP-HPLC.

Each peptide was found to coelute with its enantiomer, as expected, buteach enantiomeric pair was shown to have baseline separation (>2.0minutes) from the other enantiomer pair. Therefore, an aliquot of theGGFL-NH₂ -resin was treated with sodium hydride in dimethylsulfoxide toform the amide anions, and then quenched by washing with 1 percentwater/dimethylsulfoxide. Following cleavage from the resin, the maximumpercentage of the D/L, L/D enantiomeric pair, as seen by RP-HPLC, wasless than 0.75 percent, establishing that the extent of racemization,and therefore potential C.sub.α -methylation, was less than 1 percent.

EXAMPLE 10 Enzymatic Susceptibility

The stability of N-permethylated compounds to proteolysis was examinedfor two permethylated sequences, AlaGlyGlyPheLeu-NH₂ (SEQ ID NO:37) andArgGlyGlyPheLeu-NH₂ (SEQ ID NO:38) with their non-permethylatedequivalents used as controls. Treatment of these found compounds bytrypsin and chymotrypsin was monitored by RP-HPLC and mass spectralanalysis. Rapid cleavage of AGGFL-NH₂ (SEQ ID NO:37) by chymotrypsin andRGGFL-NH₂ (SEQ ID NO:38) by trypsin was observed (<l hour), whereas lessthan 1 percent cleavage of the equivalent permethylated peptides wasseen following overnight enzyme exposure. These results confirmed theexpectation that N-alkylated amides would be much less prone toenzymatic degradation Simon et al., Proc Natl. Acad. Sci. USA,89:9367-9371 (1992)!.

The assays were performed in 1 ml O.1M NH₄ HCO₃, pH 7.8, at roomtemperature for 16 hours at a peptide concentration of 1.0 mg/ml. Enzymeto peptide concentration was 1:50.

EXAMPLE 11 Use of a Trityl Chloride N-Terminal Blocking Group

The syntheses of this and the following examples utilize a dipeptide forsimplicity and ease of understanding. It should be understood, however,that the reactions disclosed are similarly useful on oligopeptidemixture sets, and particularly oligopeptide mixture sets of a lengthgreater than two residues.

A polypropylene mash packet containing 100 mg of Phe-Leu-solid supportmethylbenzhydralamine (MBHA) resin; 0.7202 mmol/g primary amino groups!was washed with DGM (1×1 minute×5 ml), 5 percent DIEA/DCM (3×2 minutes×5ml) and DCM (2×1 minute×5 ml). Thereafter, 3.6 ml DCM and 0.364 ml DIEA(2.0886 mmol) were added to the peptide-resin, followed by addition of100.4 mg trityl chloride (TrtCl) (0.3601 mmol). The reaction mixture wasshaken for one hour. The peptide-resin was washed with DMF (2×1 minute×5ml), 5 percent DIEA/DCM (1×2 minute×5 ml), DCM (3×1 minute×5 ml) andMeOH (1×1 minute×5 ml). A small sample of peptide-resin was assayed forremaining free amino groups using the bromophenol blue test Krchnak etal., Collect. Czech. Chem. Commun., 53:2542-2548 (1988)!. If furtherTrtCl coupling was necessary, a solvent mixture of DCM/DMF 9:1 or 8:2was used. The formed Trt-Phe-Leu-MBHA resin was dried under high vacuum(lyophilizer).

All manipulations were performed under nitrogen atmosphere and anhydrousconditions. Lithium tertbutoxide in THF (5.76 m. 0.5M; 2.8808 mmol) wasadded to Trt-Phe-Leu-MBHA resin contained in a polypropylene mash packet(0.07202 mmol peptide, 0.14404 mmol amide groups) and shaken at roomtemperature for 15 minutes. The base solution was removed by syringe.5.76 ml DMSO and the alkylation reagent, here 0.538 ml iodomethane(8.6424 mmol), were added. The reaction mixture was shaken at roomtemperature for 15 minutes. Using alkylation reagents such as allylbromide or benzyl bromide, reaction time was two hours. Thepeptide-resin was washed with DMF (3×1 minute×5 ml), IPA (2×1 minute×5ml), DCM (3×1 minute×5 ml) and MeOH (1×1 minute×5 ml) and dried underhigh vacuum (lyophilizer). The whole alkylation procedure was repeated.

The alkylated N-trityl-blocked peptide-resin was washed with DCM (1×1minute×5 ml) and then treated with 2 percent TFA in DCM (1×2 minutes×5ml and 1×30 minutes×5 ml), followed by the 1 minute washing steps DCM(1×5 ml), IPA (2×5 ml), DCM (2×5 ml) and MeOH (1×5 ml). The resulting,deblocked peptide was then ready for N-terminal alkylation or for use asa free amine. The resulting support-linked peptide can be referred to asPhe-(NMe)Leu-(NMe)-MBHA resin.

EXAMPLE 12 N-Terminal Reductive Alkylation

The Phe-(NMe)Leu-(NMe)-MBHA resin (0.07202 mmol primary amine) preparedin Example 11 was in a polypropylene mash packet and washed with DCM(1×1 minute×5 ml), 5 percent DIEA/DCM (3×2 minutes x 5 ml) and DCM (2×1minute×5 ml). Thereafter, 8.6 DCM and 0.1455 ml DIEA (0.835 mmol) wereadded to the peptide-resin, followed by addition of 37.825 mg of4,4'-dimethoxydiphenylmethyl chloride (DodCl) (0.14404 mmol). Themixture was shaken for 30 minutes. The peptide-resin was washed with DMF(2×1 minute×5 ml), 5 percent DIEA/DCM (1×2 minutes×5 ml) and DCM (3×1minute×5 ml). A sample of peptide-resin was tested for remaining freeamino groups using the bromophenol blue test. If necessary the reactionwas repeated with prolonged coupling (1 hour). The blocked,support-linked peptide so prepared can be referred to asDod-Phe-(NMe)Leu(NMe)-MBHA resin.

Formaldehyde (10 ml, 37 percent wt. solution in water), 90 ml DMF and 30g of anhydrous magnesium sulfate were mixed and shaken overnight,centrifuged, and the supernatant used for the reductive methylation asfollows. The thus prepared formaldehyde solution (8.5 ml) was added toDod-Phe-(NMe)Leu-(NMe)-MBHA resin (0.07202 mmol peptide), contained in apolypropylene mesh packet, and shaken for 5 minutes. The solution waspoured off. Additional 8.5 ml formaldehyde solution with 0.085 ml aceticacid were added and shaken. After 5 minutes, 85 mg of sodiumcyanoborohydride were added and the mixture shaken for one hour. Thepeptide-resin was washed with DMF (3×1 minute×5 ml), IPA (2×1 minute×5ml), DCM (3×1 minute×5 ml) and MeOH (1×1 minute×5 ml). The blocked,N-alkylated (N-methylated) support-linked peptide so prepared can bereferred to as Me-Dod-Phe-(NMe)Leu-(NMe)-MBHA resin.

The peptide-resin was washed with DCM (1×1 minute×5 ml), then treatedwith 55 percent TFA in DCM (1×5 minutes×5 ml and 1×30 minutes×5 ml),followed by the 1 minute washing steps DCM (1×5 ml), IPA (2×5 ml) andDCM (2×1 ml). The peptide resin so formed can be referred to asMe-Phe-(NMe)Leu-(NMe)-MBHA resin.

It should be apparent that any of the other contemplated C₂ -C₇ -alkylgroups could replace methyl in the above reductive alkylation by use ofthe appropriate aldehyde or ketone. The N-terminal monoalkylated peptideor library of peptides that results from the above procedures can beused individually as is, used as mixtures when so prepared, or can belinked to another amino acid residue, and the chain elongated.

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications may be effectedwithout departing from the true spirit and scope of the novel conceptsof the invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 38                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CysGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AspGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GluGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       PheGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GlyGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       HisGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       IleGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       LysGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      LeuGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa represents methionine                      sulfoxide."                                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      XaaGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AsnGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ProGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GlnGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ArgGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      SerGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ThrGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ValGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TrpGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a peralkylated                         peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TyrGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      PhePhePhePhe                                                                  (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      PhePhePhePhePhe                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      PhePhePhePhePhePhe                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..7                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      PhePhePhePhePhePhePhe                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..8                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      PhePhePhePhePhePhePhePhe                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      LeuPheIlePhePhePhe                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      PhePheIlePhePhePhe                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      LeuPhePhePhePhePhe                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      PhePhePhePheHisPhe                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      LeuPheIlePhePheHis                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      LeuPhePhePheHisPhe                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      LeuPheIlePheHisPhe                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      LeuPhePhePhePheHis                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      PhePheIlePhePheHis                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide. Xaa in the first position can be either                              trimethyl ammonium Leu or trimethyl ammonium Phe; Xaa                         in the third position can be either His or Phe; Xaa in                        the fifth position can be either Ile or Phe; and, Xaa in                      the sixth position can be either methyl carboxyamido His                      or methyl carboxyamido Phe. At least one of Xaa in the                        fifth or sixth position is Phe."                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      XaaPheXaaPheXaaXaa                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GlyGlyPheLeu                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This is a permethylated                        peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      AlaGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "This a permethylated                           peptide."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      ArgGlyGlyPheLeu                                                               15                                                                            __________________________________________________________________________

We claim:
 1. A process for determining the sequence of a linearperalkylated oligopeptide that preferentially binds to an acceptor thatcomprises the steps of:(a) providing a library of sets of linearperalkylated oligopeptides in which each set comprises a mixture ofequimolar amounts of linear C₁ -C₇ -alkyl peralkylated oligopeptidemember chains containing the same number of two to about tenperalkylated amino acid residues in lack peralkylated oligopeptidechain, each peralkylated amino acid residue except proline having itspeptidyl amido nitrogen atom alkylated with a C₁ -C₇ -alkyl group, themember chains of each set having one or more of at least six differentpredetermined peralkylated amino acid residues at the same one or morepredetermined positions of the peralkylated oligopeptide chain, and eachset having an equimolar amount of at least six different peralkylatedamino acid residues at the same one or more other positions of theperalkylated oligopeptide chain, the amino-terminus of each peralkylatedoligopeptide being selected from the group consisting of a quaternary C₁-C₇ -alkylammonium group, an amino group, an N--C₁ -C₇ -alkylamino, anN--C₁ -C₇ -alkyl-N--C₁ -C₁₈ hydrocarboyl and a pyroglutamoyl group, andthe carboxy-terminus being selected from the group consisting of a C₁-C₇ -alkyl carboxylic ester, mono- or di-N--C₁ -C₇ -alkylcarboxamide anda carboxyl group, the sets differing in that the one or morepredetermined peralkylated amino acid residue present at the same one ormore predetermined chain positions within each set are different betweenthe sets; (b) separately admixing each set from said library of setswith said acceptor in an aqueous medium at a set concentration of about0.1 milligrams per liter to about 100 grams per liter, separatelyassaying the binding of each set to the acceptor, and determining a setexhibiting preferential, specific binding relative to the other sets,thereby identifying a peralkylated amino acid residue that providedpreferential binding at said one or more predetermined positions; (c)providing a second library of sets of linear C₁ -C₇ -alkyl peralkylatedoligopeptides in which each set comprises a mixture of equimolar amountsof member linear peralkylated oligopeptide chains containing the samenumber of two to about ten peralkylated amino acid residues in eachperalkylated oligopeptide chain as the chains of the first-named libraryof sets, each peralkylated amino acid residue except proline having itspeptidyl amido nitrogen atom alkylated with a C₁ -C₇ -alkyl group, themember chains of each second library of sets containing the one or moreperalkylated amino acid residues of the first-named set identified asexhibiting preferential, specific binding in the same one or morepredetermined chain positions in said first-named sets, the memberchains of said second sets having a predetermined one of said at leastsix different peralkylated amino acid side chains at anotherpredetermined position of the peralkylated oligopeptide chain differentfrom said one or more positions of the identified peralkylated aminoacid residue of the first-named library of sets, each of said secondlibrary of sets having equimolar amounts of said at least six differentperalkylated amino acid residues of said first-named sets at the sameone or more positions of the peralkylated oligopeptide chain notoccupied by said one or more identified peralkylated amino acid residueor the predetermined peralkylated amino acid residues, having one fewerperalkylated oligopeptide positions occupied by equimolar amounts of atleast six different peralkylated amino acid residues, and having thesame amino- and carboxy-termini as the peralkylated oligopeptides ofsaid first-named set; (d) separately admixing each set of said secondlibrary of sets with said acceptor in an aqueous medium at a setconcentration of about 0.1 milligrams per liter to about 100 grams perliter, separately assaying the binding of each set to the acceptor, anddetermining a second set exhibiting preferential, specific binding,thereby identifying a peralkylated amino acid side chain that providespreferential binding at said other predetermined position in theperalkylated oligopeptide chain; (e) repeating steps (c) and (d) usingzero through seven further libraries of sets of linear peralkylatedoligopeptides instead of said second plurality of sets or untilpreferential, specific binding does not increase when a further libraryis assayed, each further library of sets of linear C₁ -C₇ -alkylperalkylated oligopeptides comprising a mixture of equimolar amounts ofmember linear peralkylated oligopeptide chains containing the samenumber of two to about ten peralkylated amino acid residues in eachperalkylated oligopeptide chain as the chains of the first-namedplurality of sets, the member chains of the sets of each further librarycontaining the peralkylated amino acid residues in the peralkylatedoligopeptide chain positions that exhibited preferential binding in alibrary of sets used immediately before, and a predetermined one of saidat least six different peralkylated amino acid residues at anotherpredetermined position of the peralkylated oligopeptide chain differentfrom the positions of the identified peralkylated amino acid residues ofthe library of sets used immediately before, each of said furtherlibrary of sets having equimolar amounts of said at least six differentperalkylated amino acid residues of said first-named sets at the sameone or more positions of the peralkylated oligopeptide chain notoccupied by the identified peralkylated amino acid residues or thepredetermined peralkylated amino acid residues, and having the sameamino- and carboxy-termini as the peralkylated oligopeptides of saidfirst-named set; (f) where the last-assayed library of sets exhibitsincreased preferential, specific binding compared to the library usedimmediately before and one position of the peralkylated oligopeptidechain that provides preferential, specific binding is not identified,providing at least six peralkylated oligopeptide chains in which eachchain contains the same number of two to about ten peralkylated aminoacid residues in each peralkylated oligopeptide chain as the chains ofthe first-named plurality of sets, each peralkylated oligopeptide chaincontaining the identified peralkylated amino acid residues in theperalkylated oligopeptide chain positions that exhibited increasedpreferential, specific binding in the immediately preceding assay ofstep (e) and a predetermined one of said at least six differentperalkylated amino acid residues at another predetermined position inthe peralkylated oligopeptide chain different from the positions of theidentified peralkylated amino acid residues used in the immediatelypreceding assay of step (e), and having the same amino- andcarboxy-termini as the peralkylated oligopeptides of said first-namedset; and (g) separately admixing each of said at least six peralkylatedoligopeptides of step (f) with said acceptor in an aqueous medium at aperalkylated oligopeptide concentration of about 0.1 milligrams to about100 grams per liter, separately assaying the binding of eachperalkylated oligopeptide, and determining the peralkylated oligopeptideexhibiting preferential, specific binding, thereby determining thesequence of a linear peralkylated oligopeptide that preferentially bindsto said acceptor.
 2. The process according to claim 1 wherein saididentified and predetermined peralkylated amino acid residues areadjacent peralkylated residues.
 3. The process according to claim 2wherein said predetermined one or more of said at least six peralkylatedamino acid residues at one or more predetermined positions of (a)include a terminal position of the peralkylated oligopeptide chain. 4.The process according to claim 2 wherein said first-named peralkylatedoligopeptide chains contain about five to about eight peralkylated aminoacid residues.
 5. The process according to claim 2 wherein at least tendifferent peralkylated amino acid residues are utilized instead of atleast six.
 6. The process according to claim 2 wherein saidpredetermined one or more of at least six different peralkylated aminoacid residues at one or more predetermined positions of the sets of (a)are the same as said at least six different peralkylated amino acidresidues present in equimolar amounts.
 7. The process according to claim1 wherein said acceptor is a cellular receptor.
 8. The process accordingto claim 7 wherein said cellular receptor is present in a bacterium oryeast cell cultured in a growth medium.
 9. The process according toclaim 7 wherein each peralkylated oligopeptide set is provided coupledto a solid support.
 10. A process for determining the sequence of alinear peralkylated oligopeptide that preferentially binds to anacceptor that comprises the steps of:(a) providing separate pluralitiesof sets of linear peralkylated oligopeptides, each set of thosepluralities comprising a mixture of equimolar amounts of linear C₁ -C₇-alkyl peralkylated linear oligopeptide chains containing the samenumber of two to about ten peralkylated amino acid residues in eachchain, each peralkylated amino acid residue except proline having itspeptidyl amido nitrogen atom alkylated with a C₁ -C₇ -alkyl group, eachset of peralkylated oligopeptide chains having a single one of at leastsix different predetermined peralkylated amino acid residues at a singlepredetermined position of the peralkylated oligopeptide chain, and eachset having equimolar amounts of each of said at least six differentperalkylated amino acid residues at the other positions of theperalkylated oligopeptide chain, each set differing from the other setsin the identity and chain position of said single predeterminedperalkylated amino acid residue present at the predetermined repeatingunit position within the set, the amino-terminus of each oligopeptidebeing selected from the group consisting of a quaternary C₁ -C₇-alkylammonium group, an amino group, an N--C₁ -C₇ -alkylamino, an N--C₁-C₇ -alkyl-N--C₁ -C₁₈ hydrocarboyl and a pyroglutamoyl group, and thecarboxy-terminus being selected from the group consisting of a C₁ -C₇-alkyl carboxylic ester, mono- or di-N--C₁ -C₇ -alkylcarboxamide and acarboxyl group; (b) separately admixing each set with said acceptor inan aqueous medium at a set concentration of about 0.1 milligrams perliter to about 100 grams per liter, and separately assaying the bindingof each set to the acceptor;a peralkylated amino acid residue thatexhibited preferential, specific binding at each peralkylated positionof the peralkylated oligopeptide chain of each set providing a sequenceof a peralkylated linear oligopeptide that preferentially binds to saidacceptor.
 11. The process according to claim 10 wherein the single,predetermined positions of the plurality of sets, taken as a group, areadjacent to each other in the peralkylated oligopeptide chain.
 12. Theprocess according to claim 10 wherein each peralkylated oligopeptidechain contains about five to about eight peralkylated amino acidresidues.
 13. The process according to claim 10 wherein said singlepredetermined peralkylated amino acid residue of each peralkylatedoligopeptide chain is one of at least ten different peralkylated aminoacid residues, and the same at least ten different peralkylated aminoacid residues are present in equimolar amounts at the other peralkylatedoligopeptide positions of each set.
 14. The process according to claim10 wherein each of said sets is provided not coupled to a solid supportused for synthesis.